(en)Transgenic seed for crops with enhanced agronomic traits are provided by trait-improving recombinant DNA in the nucleus of cells of the seed where plants grown from such transgenic seed exhibit one or more enhanced traits as compared to a control plant. Of particular interest are transgenic plants that have increased yield. The present invention also provides recombinant DNA molecules for expression of a protein, and recombinant DNA molecules for suppression of a protein.

1.ApplicationNumber: US-29005708-A
1.PublishNumber: US-2009144847-A1
2.Date Publish: 20090604
3.Inventor: SHAIKH FATEN
COFFIN MARIE A.
BOHANNON MARIA C.
WINSLOW AMANDA

4.Inventor Harmonized: SHAIKH FATEN(US)
COFFIN MARIE A(US)
BOHANNON MARIA C(US)
WINSLOW AMANDA(US)

5.Country: US
6.Claims:
(en)Transgenic seed for crops with enhanced agronomic traits are provided by trait-improving recombinant DNA in the nucleus of cells of the seed where plants grown from such transgenic seed exhibit one or more enhanced traits as compared to a control plant. Of particular interest are transgenic plants that have increased yield. The present invention also provides recombinant DNA molecules for expression of a protein, and recombinant DNA molecules for suppression of a protein.

7.Description:
(en)CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit under 35 U.S.C. § 119(e) of U.S. provisional application Ser. No. 61/190,041 filed on Oct. 30, 2007 which is incorporated herein by reference in its entirety.


INCORPORATION OF SEQUENCE LISTING
Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form (CRF) of the sequence listing, all on CD-Rs, each containing the file named 38-21(54975)B_seqListing.txt, which is 12,914,688 bytes (measured in MS-WINDOWS) and was created on Oct. 23, 2008, are incorporated herein by reference in their entirety.
INCORPORATION OF COMPUTER PROGRAM LISTING
A Computer Program Listing with folders “hmmer-2.3.2” and “47pfamDir” is contained on a CD-R and is incorporated herein by reference in their entirety. Folder hmmer-2.3.2 contains the source code and other associated file for implementing the HMMer software for Pfam analysis. Folder 47pfamDir contains 47 profile Hidden Markov Models. Both folders were created on the disk on Oct. 23, 2008 having a total size of 4,816,896 bytes when measured in MS-WINDOWS® operating system.
FIELD OF THE INVENTION
Disclosed herein are transgenic plant cells, plants and seeds comprising recombinant DNA and methods of making and using such plant cells, plants and seeds.
SUMMARY OF THE INVENTION
This invention provides plant cell nuclei with recombinant DNA that imparts enhanced agronomic traits in transgenic plants having the nuclei in their cells, e.g. enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced shade tolerance, enhanced high salinity tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil. In certain cases the trait is imparted by producing in the cells a protein that is encoded by recombinant DNA and/or in other cases the trait is imparted by suppressing the production of a protein that is natively produced in the cells.
Such recombinant DNA in a plant cell nucleus of this invention is provided in as a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein or to DNA that results in gene suppression. Such DNA in the construct is sometimes defined by protein domains of an encoded protein targeted for production or suppression, e.g. a “Pfam domain module” (as defined herein below) from the group of Pfam domain modules identified in Table 17. Alternatively, e.g. where a Pfam domain module is not available, such DNA in the construct is defined a consensus amino acid sequence of an encoded protein that is targeted for production or suppression, e.g. a protein having amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of SEQ ID NO: 4819 through SEQ ID NO: 4825.
Other aspects of the invention are directed to specific derivative physical forms of the transgenic plant cell nuclei, e.g. where such a transgenic nucleus is present in a transgenic plant cell, a transgenic plant including plant part(s) such as progeny transgenic seed, and a haploid reproductive derivative of plant cell such as transgenic pollen and transgenic ovule. Such plant cell nuclei and derivatives are advantageously selected from a population of transgenic plants regenerated from plant cells having a nucleus that is transformed with recombinant DNA by screening the transgenic plants or progeny seeds in the population for an enhanced trait as compared to control plants or seed that do not have the recombinant DNA in their nuclei, where the enhanced trait is enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced shade tolerance, enhanced high salinity tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil.
In other aspects of the invention the nuclei of plant cells and derivative transgenic cells, plants, seeds, pollen and ovules further include recombinant DNA expressing a protein that provides tolerance from exposure to one or more herbicide applied at levels that are lethal to a wild type plant. Such herbicide tolerance is not only an advantageous trait in such plants but is also useful as a selectable marker in the transformation methods for producing the nuclei and nuclei derivatives of the invention. Such herbicide tolerance includes tolerance to a glyphosate, dicamba, or glufosinate herbicide.
Yet other aspects of the invention provide transgenic plant cell nuclei which are homozygous for the recombinant DNA. The transgenic plant cell nuclei of the invention and derivative cells, plants, seed and haploid reproductive derivatives of the invention are advantageously provided in corn, soybean, cotton, canola, alfalfa, wheat, rice plants, or combinations thereof.
This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA in the nucleus of the plant cells. More specifically the method includes, but are not limited to, (a) screening a population of plants for an enhanced trait and recombinant DNA, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants which do not express the recombinant DNA; (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants and (c) collecting seed from a selected plant. Such method can further include the steps of (a) verifying that the recombinant DNA is stably integrated in said selected plants; and (b) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by a recombinant DNA with a sequence of one of SEQ ID NO: 1-114; In one aspect of the invention the plants in the population can further include DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells and where the selecting is effected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the invention, the plants are selected by identifying plants with the enhanced trait. The methods can be used for manufacturing corn, soybean, cotton, canola, alfalfa, wheat and/or rice seed selected as having one of the enhanced traits described above.
Another aspect of the invention provides a method of producing hybrid corn seed including the step of acquiring hybrid corn seed from a herbicide tolerant corn plant which also has a nucleus of this invention with stably-integrated, recombinant DNA. The method can further include the steps of producing corn plants from said hybrid corn seed, where a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and/or a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1 , 2 and 3 are pictures illustrating plasmid maps.
FIG. 4 illustrates a consensus amino acid sequence of SEQ ID NO: 181 and its homologs.



DETAILED DESCRIPTION OF THE INVENTION
In the attached sequence listing:
SEQ ID NO: 1-114 are nucleotide sequences of the coding strand of DNA for “genes” used in the recombinant DNA imparting an enhanced trait in plant cells, i.e. each represents a coding sequence for a protein;
SEQ ID NO: 115-228 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequence 1-114;
SEQ ID NO: 229-4815 are amino acid sequences of homologous proteins;
SEQ ID NO: 4816 is a nucleotide sequence of a plasmid base vector useful for corn transformation; and
SEQ ID NO: 4817 is a DNA sequence of a plasmid base vector useful for soybean transformation.
SEQ ID NO: 4818 is a DNA sequence of a plasmid base vector useful for cotton transformation.
SEQ ID NO: 4819-4825 are consensus sequences.
Table 1 lists the protein SEQ ID NOs and their corresponding consensus SEQ ID NOs.









TABLE 1





GENE ID
PEP SEQ ID
CONSENSUS SEQ ID





CGPG3454
151
4819


CGPG4528
181
4820


CGPG4560
183
4821


CGPG7500
206
4822


CGPG7639
213
4823


CGPG8137
226
4824


CGPG8180
228
4825
























The nuclei of this invention are identified by screening transgenic plants for one or more traits including enhanced drought stress tolerance, enhanced heat stress tolerance, enhanced cold stress tolerance, enhanced high salinity stress tolerance, enhanced low nitrogen availability stress tolerance, enhanced shade stress tolerance, enhanced plant growth and development at the stages of seed imbibition through early vegetative phase, and enhanced plant growth and development at the stages of leaf development, flower production and seed maturity.
“Gene” means a chromosomal element for expressing a protein and specifaccly includes the DNA encoding a protein. In cases where expression of a target protein is desired, the pertinent part of a gene is the DNA encoding the target protein; in cases where suppression of a target is desired, the pertinent part of a gene is that part that is transcribed as mRNA. “Recombinant DNA” means a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous elements in a transcription unit. Recombinant DNA can include DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form.
“Trait” means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell, or any combinations thereof.
A “control plant” is a plant without trait-improving recombinant DNA in its nucleus. A control plant is used to measure and compare trait enhancement in a transgenic plant with such trait-improving recombinant DNA. A suitable control plant can be a non-transgenic plant of the parental line used to generate a transgenic plant herein. Alternatively, a control plant can be a transgenic plant having an empty vector or marker gene, but does not contain the recombinant DNA that produces the trait enhancement. A control plant can also be a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait enhancement, the use of a limited number of control plants can cause a wide variation in the control dataset. To minimize the effect of the variation within the control dataset, a “reference” is used. As use herein a “reference” is a trimmed mean of all data from both transgenic and control plants grown under the same conditions and at the same developmental stage. The trimmed mean is calculated by eliminating a specific percentage, i.e., 20%, of the smallest and largest observation from the data set and then calculating the average of the remaining observation.
“Trait enhancement” means a detectable and desirable difference in a characteristic in a transgenic plant relative to a control plant or a reference. In some cases, the trait enhancement can be measured quantitatively. For example, the trait enhancement can entail at least a 2% desirable difference in an observed trait, at least a 5% desirable difference, at least about a 10% desirable difference, at least about a 20% desirable difference, at least about a 30% desirable difference, at least about a 50% desirable difference, at least about a 70% desirable difference, or at least about a 100% difference, or an even greater desirable difference. In other cases, the trait enhancement is only measured qualitatively. It is known that there can be a natural variation in a trait. Therefore, the trait enhancement observed entails a change of the normal distribution of the trait in the transgenic plant compared with the trait distribution observed in a control plant or a reference, which is evaluated by statistical methods provided herein. Trait enhancement includes, but is not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions can include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability, high plant density, or any combinations thereof.
Many agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, juvenile traits, or any combinations thereof. Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Also of interest is the generation of transgenic plants that demonstrate desirable phenotypic properties that can confer an increase in overall plant yield. Such properties include enhanced plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.
“Yield-limiting environment” means the condition under which a plant would have the limitation on yield including environmental stress conditions.
“Stress condition” means a condition unfavorable for a plant, which adversely affect plant metabolism, growth and/or development. A plant under the stress condition typically shows reduced germination rate, retarded growth and development, reduced photosynthesis rate, and eventually leading to reduction in yield. Specifically, “water deficit stress” used herein refers to the sub-optimal conditions for water and humidity needed for normal growth of natural plants. Relative water content (RWC) can be used as a physiological measure of plant water deficit. It measures the effect of osmotic adjustment in plant water status, when a plant is under stressed conditions. Conditions which can result in water deficit stress include, but are not limited to, heat, drought, high salinity and PEG induced osmotic stress.
“Cold stress” means the exposure of a plant to a temperatures below (two or more degrees Celsius below) those normal for a particular species or particular strain of plant.
“Nitrogen nutrient” means any one or any mix of the nitrate salts commonly used as plant nitrogen fertilizer, including, but not limited to, potassium nitrate, calcium nitrate, sodium nitrate, ammonium nitrate. The term ammonium as used herein means any one or any mix of the ammonium salts commonly used as plant nitrogen fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium sulfate, etc.
“Low nitrogen availability stress” means a plant growth condition that does not contain sufficient nitrogen nutrient to maintain a healthy plant growth and/or for a plant to reach its typical yield under a sufficient nitrogen growth condition. For example, a limiting nitrogen condition can refers to a growth condition with 50% or less of the conventional nitrogen inputs. “Sufficient nitrogen growth condition” means a growth condition where the soil or growth medium contains or receives optimal amounts of nitrogen nutrient to sustain a healthy plant growth and/or for a plant to reach its typical yield for a particular plant species or a particular strain. One skilled in the art would recognize what constitute such soil, media and fertilizer inputs for most plant species.
“Shade stress” means a growth condition that has limited light availability that triggers the shade avoidance response in plant. Plants are subject to shade stress when localized at lower part of the canopy, or in close proximity of neighboring vegetation. Shade stress can become exacerbated when the planting density exceeds the average prevailing density for a particular plant species.
“Increased yield” of a transgenic plant of the present invention is evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e., seeds, or weight of seeds, per acre), bushels per acre, tons per acre, tons per acre, kilo per hectare. For example, maize yield can be measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture. Increased yield can result from enhanced utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from enhanced tolerance to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Trait-improving recombinant DNA can also be used to provide transgenic plants having enhanced growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.
A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which include genes expressed in plant cells such Agrobacterium or Rhizobium . Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissues are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that can effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions.
As used herein, “operably linked” refers to the association of two or more nucleic acid elements in a recombinant DNA construct, e.g. as when a promoter is operably linked with DNA that is transcribed to RNA whether for expressing or suppressing a protein. Recombinant DNA constructs can be designed to express a protein which can be an endogenous protein, an exogenous homologue of an endogenous protein or an exogenous protein with no native homologue. Alternatively, recombinant DNA constructs can be designed to suppress the level of an endogenous protein, e.g. by suppression of the native gene. Such gene suppression can be effectively employed through a native RNA interference (RNAi) mechanism in which recombinant DNA comprises both sense and anti-sense oriented DNA matched to the gene targeted for suppression where the recombinant DNA is transcribed into RNA that can form a double-strand to initiate an RNAi mechanism. Gene suppression can also be effected by recombinant DNA that comprises anti-sense oriented DNA matched to the gene targeted for suppression. Gene suppression can also be effected by recombinant DNA that comprises DNA that is transcribed to a microRNA matched to the gene targeted for suppression. In the examples illustrating the invention recombinant DNA for effecting gene suppression that imparts is identified by the term “antisense”. It will be understood by a person of ordinary skill in the art that any of the ways of effecting gene suppression are contemplated and enabled by a showing of one approach to gene suppression.
A “consensus amino acid sequence” means an artificial, amino acid sequence indicating conserved amino acids in the sequence of homologous proteins as determined by statistical analyis of an optimal alignment, e.g. CLUSTALW, of amino acid sequence of homolog proteins. The consensus sequences listed in the sequence listing were created by identifying the most frequent amino acid at each position in a set of aligned protein sequences. When there was 100% identity in an alignment the amino acid is indicated by a capital letter. When the occurance of an amino acid is at least about 70% in an alignment, the amino acid is indicated by a lower case letter. When there is no amino acid occurance of at least about 70%, e.g. due to diversity or gaps, the amino acid is indicated by an “x”. When used to defined embodiments of the invention, a consensus amino acid sequence will be aligned with a query protein amino acid sequence in an optimal alignment, e.g. CLUSTALW. An embodiment of the invention will have identity to the conserved amino acids indicated in the consensus amino acid sequence.
As used herein a “homolog” means a protein in a group of proteins that perform the same biological function, e.g. proteins that belong to the same Pfam protein family and that provide a common enhanced trait in transgenic plants of this invention. Homologs are expressed by homologous genes. With reference to homologous genes, homologs include orthologs, i.e. genes expressed in different species that evolved from a common ancestral genes by speciation and encode proteins retain the same function, but do not include paralogs, i.e. genes that are related by duplication but have evolved to encode proteins with different functions. Homologous genes include naturally occurring alleles and artificially-created variants. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. When optimally aligned, homolog proteins have typically at least about 60% identity, in some instances at least about 70%, for example about 80% and even at least about 90% identity over the full length of a protein identified as being associated with imparting an enhanced trait when expressed in plant cells. In one aspect of the invention homolog proteins have an amino acid sequence that has at least 90% identity to a consensus amino acid sequence of proteins and homologs disclosed herein.
Homologs are identified by comparison of amino acid sequence, e.g. manually or by use of a computer-based tool using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. A local sequence alignment program, e.g. BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. Because a protein hit with the best E-value for a particular organism may not necessarily be an ortholog, i.e. have the same function, or be the only ortholog, a reciprocal query is used to filter hit sequences with significant E-values for ortholog identification. The reciprocal query entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit can be identified as an ortholog, when the reciprocal query's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. A further aspect of the homologs encoded by DNA useful in the transgenic plants of the invention are those proteins that differ from a disclosed protein as the result of deletion or insertion of one or more amino acids in a native sequence.
As used herein, “percent identity” means the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, for example nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. “Percent identity” (“% identity”) is the identity fraction times 100. Such optimal alignment is understood to be deemed as local alignment of DNA sequences. For protein alignment, a local alignment of protein sequences should allow introduction of gaps to achieve optimal alignment. Percent identity is calculated over the aligned length not including the gaps introduced by the alignment per se.
Homologous genes are genes which encode proteins with the same or similar biological function to the protein encoded by the second gene. Homologous genes can be generated by the event of speciation (see ortholog) or by the event of genetic duplication (see paralog). “Orthologs” refer to a set of homologous genes in different species that evolved from a common ancestral gene by specification. Normally, orthologs retain the same function in the course of evolution; and “paralogs” refer to a set of homologous genes in the same species that have diverged from each other as a consequence of genetic duplication. Thus, homologous genes can be from the same or a different organism. As used herein, “homolog” means a protein that performs the same biological function as a second protein including those identified by sequence identity search.
“ Arabidopsis ” means plants of Arabidopsis thaliana.
“Pfam” database is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 19.0 (December 2005) contains alignments and models for 8183 protein families and is based on the Swissprot 47.0 and SP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. The Pfam database is currently maintained and updated by the Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the protein family alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low.
A “Pfam domain module” is a representation of Pfam domains in a protein, in order from N terminus to C terminus. In a Pfam domain module individual Pfam domains are separated by double colons “::”. The order and copy number of the Pfam domains from N to C terminus are attributes of a Pfam domain module. Although the copy number of repetitive domains is important, varying copy number often enables a similar function. Thus, a Pfam domain module with multiple copies of a domain should define an equivalent Pfam domain module with variance in the number of multiple copies. A Pfam domain module is not specific for distance between adjacent domains, but contemplates natural distances and variations in distance that provide equivalent function. The Pfam database contains both narrowly- and broadly-defined domains, leading to identification of overlapping domains on some proteins. A Pfam domain module is characterized by non-overlapping domains. Where there is overlap, the domain having a function that is more closely associated with the function of the protein (based on the E value of the Pfam match) is selected.
Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins with the same Pfam domain module are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Models which characterizes the Pfam domains using HMMER software, a current version of which is provided in the appended computer listing. Candidate proteins meeting the same Pfam domain module are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein with a common Pfam domain module for recombinant DNA in the plant cells of this invention are also included in the appended computer listing.
Version 19.0 of the HMMER software and Pfam databases were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 115 through SEQ ID NO:228. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 19 by Pfam analysis disclosed herein can be used in recombinant DNA of the plant cells of this invention, e.g. for selecting transgenic plants having enhanced agronomic traits. The relevant Pfam modules for use in this invention, as more specifically disclosed below, are Saccharop_dh, Isoamylase_AP2, zf-C2H2, PLATZ, F-box::Tub, zf-C3HC4::YDG_SRA::zf-C3HC4, SBP, HLH, AP2, zf-B_box::zf-B_box, zf-C3HC4, AP2, HMG_box::HMG_box::HMG_box, zf-C3HC4, zf-C2H2, GATA, HLH, AP2, NAM, zf-Dof, WRKY, AP2, HMG_box, zf-CCCH::KH — 1::zf-CCCH, SRF-TF, WRKY, zf-C3HC4, zf-Dof, zf-Dof, AP2, AP2, DUF248, zf-C2H2, SRF-TF::K-box, zf-Dof, zf-C2H2, Myb_DNA-binding::Myb_DNA-binding, Myb_DNA-binding::Myb_DNA-binding::Myb_DNA-binding, SRF-TF, Pex2_Pex12::zf-C3HC4, bZIP — 2, HLH, GRAS, Myb_DNA-binding, GRAS, F-box, GRAS, WRKY, AT_hook::DUF296, GRAS, Myb_DNA-binding::Myb_DNA-binding, Myb_DNA-binding::Myb_DNA-binding, HLH, zf-C2H2, NAM, zf-B_box::zf-B_box, Myb_DNA-binding, NAM, PHD, SRF-TF::K-box, zf-C3HC4, HLH, SRF-TF::K-box, Myb_DNA-binding, WRVKY, SRF-TF::K-box, Myb_DNA-binding, Myb_DNA-binding::Linker_histone, Myb_DNA-binding::Myb_DNA-binding, Myb_DNA-binding::Myb_DNA-binding, bZIP — 1, Myb_DNA-binding::Myb_DNA-binding, SRF-TF::K-box, SRF-TF::K-box, CBFD_NFYB_HMF, AUX_IAA, Myb_DNA-binding::Myb_DNA-binding, LIM::LIM, IBR, SET, bZIP — 1, Mov34, ZZ::Myb_DNA-binding::SWIRM, bZIP — 1, E2F_TDP::E2F_TDP, Myb_DNA-binding, Prefoldin, NAM, HD-ZIP_N::Homeobox::HALZ, Myb_DNA-binding::Myb_DNA-binding, AP2, GATA, zf-C3HC4, SRF-TF::K-box, bZIP — 1, TCP, zf-C3HC4, Ank::Ank::zf-CCCH::zf-CCCH, Myb_DNA-binding::Myb_DNA-binding, TCP, AP2, ZZ, zf-C2H2::zf-C2H2, AP2, SRF-TF::K-box, B3, zf-LSD1::zf-LSD1, and HLH.
Recombinant DNA Constructs
The invention uses recombinant DNA for imparting one or more enhanced traits to transgenic plant when incorporated into the nucleus of the plant cells. Such recombinant DNA is a construct comprising a promoter operatively linked to DNA for expression or suppression of a target protein in plant cells. Other construct components can include additional regulatory elements, such as 5′ or 3′ untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides. Such recombinant DNA constructs can be assembled using methods known to those of ordinary skill in the art.
Recombinant constructs prepared in accordance with the present invention also generally include a 3′ untranslated DNA region (UTR) that typically contains a polyadenylation sequence following the polynucleotide coding region. Examples of useful 3′ UTRs include those from the nopaline synthase gene of Agrobacterium tumefaciens (nos), a gene encoding the small subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacterium tumefaciens.
Constructs and vectors can also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, incorporated herein by reference.
Table 2 provides a list of genes that provided recombinant DNA that was expressed in a model plant and identified from screening as imparting an enhanced trait. When the stated orientation is “sense”, the expression of the gene or a homolog in a crop plant provides the means to identify transgenic events that provide an enhanced trait in the crop plant. When the stated orientation is “antisense”, the suppression of the native homolog in a crop plant provides the means to identify transgenic events that provide an enhanced trait in the crop plant. In some cases the expression/suppression in the model plant exhibited an enhanced trait that corresponds to an enhanced agronomic trait, e.g. cold stress tolerance, water deficit stress tolerance, low nitrogen stress tolerance and the like. In other cases the expression/suppression in the model plant exhibited an enhanced trait that is a surrogate to an enhanced agronomic trait, e.g. salinity stress tolerance being a surrogate to drought tolerance or improvement in plant growth and development being a surrogate to enhanced yield. Even when expression of a transgene or suppression of a native gene imparts an enhanced trait in a model plant, not every crop plant expressing the same transgene or suppressing the same native gene will necessarily demonstrate an indicated enhanced agronomic trait. For instance, it is well known that multiple transgenic events are required to identify a transgenic plant that can exhibit an enhanced agronomic trait. A skilled artisan can identify a transgenic plant cell nuclei, cell, plant or seed by making number of transgenic events, typically a very large number, and engaging in screening processes identified in this specification and illustrated in the examples. For example, a screening process includes selecting only those transgenic events with an intact, single copy of the recombinant DNA in a single locus of the host plant genome and further screening for transgenic events that impart a desired trait that is replicatable when the recombinant DNA is introgressed into a variety of germplams without imparting significant adverse traits.
An understanding of Table 2 is facilitated by the following description of the headings:
“NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence in the Sequence Listing.
“PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for the amino acid sequence of a protein cognate to a particular DNA
“construct_id” refers to an arbitrary number used to identify a particular recombinant DNA construct comprising the particular DNA.
“Gene ID” refers to an arbitrary name used to identify the particular DNA.
“orientation” refers to the orientation of the particular DNA in a recombinant DNA construct relative to the promoter.









TABLE 2



NUC
PEP




Seq
SEQ

Construct

ID No.
ID
Gene ID
ID
Orientation











1
115
CGPG110
10177
ANTI-SENSE

2
116
CGPG1135
12155
ANTI-SENSE

3
117
CGPG1180
12233
SENSE

4
118
CGPG1197
11873
ANTI-SENSE

5
119
CGPG1802
17308
SENSE

6
120
CGPG2561
78706
SENSE

7
121
CGPG2580
72666
SENSE

8
122
CGPG2582
17520
SENSE

9
123
CGPG2591
72672
SENSE

10
124
CGPG2597
78977
SENSE

11
125
CGPG2602
17468
SENSE

12
126
CGPG2642
75090
SENSE

13
127
CGPG2651
72018
SENSE

14
128
CGPG2662
17524
SENSE

15
129
CGPG2708
72078
SENSE

16
130
CGPG2710
72977
SENSE

17
131
CGPG2722
17445
SENSE

18
132
CGPG2736
78979
SENSE

19
133
CGPG2759
18453
SENSE

20
134
CGPG2765
17913
SENSE

21
135
CGPG2802
18210
SENSE

22
136
CGPG2823
78710
SENSE

23
137
CGPG2889
73606
SENSE

24
138
CGPG2902
18446
SENSE

25
139
CGPG2913
75011
SENSE

26
140
CGPG2945
18507
SENSE

27
141
CGPG2962
18388
SENSE

28
142
CGPG2967
18540
SENSE

29
143
CGPG2983
19653
SENSE

30
144
CGPG2996
18411
SENSE

31
145
CGPG3307
70737
SENSE

32
146
CGPG3311
18315
SENSE

33
147
CGPG3350
75080
SENSE

34
148
CGPG3355
19191
SENSE

35
149
CGPG3447
18431
SENSE

36
150
CGPG3453
18433
SENSE

37
151
CGPG3454
18434
SENSE

38
152
CGPG3455
19536
SENSE

39
153
CGPG3488
76577
SENSE

40
154
CGPG353
11113
ANTI-SENSE

41
155
CGPG3537
19614
SENSE

42
156
CGPG355
18204
SENSE

43
157
CGPG3751
73614
SENSE

44
158
CGPG3755
70450
SENSE

45
159
CGPG3756
70618
SENSE

46
160
CGPG3757
70458
SENSE

47
161
CGPG3776
71306
SENSE

48
162
CGPG3778
70451
SENSE

49
163
CGPG3799
70453
SENSE

50
164
CGPG3822
71311
SENSE

51
165
CGPG3890
76533
SENSE

52
166
CGPG3965
70955
SENSE

53
167
CGPG3966
77708
SENSE

54
168
CGPG3974
70954
SENSE

55
169
CGPG3977
19742
SENSE

56
170
CGPG3984
70960
SENSE

57
171
CGPG3988
19940
SENSE

58
172
CGPG4068
70921
SENSE

59
173
CGPG4105
70953
SENSE

60
174
CGPG4109
70978
SENSE

61
175
CGPG4125
70940
SENSE

62
176
CGPG4126
70977
SENSE

63
177
CGPG4167
70993
SENSE

64
178
CGPG4176
19762
SENSE

65
179
CGPG4193
70938
SENSE

66
180
CGPG4210
78653
SENSE

67
181
CGPG4528
73216
SENSE

68
182
CGPG4540
71139
SENSE

69
183
CGPG4560
74227
SENSE

70
184
CGPG4571
70678
SENSE

71
185
CGPG4622
70769
SENSE

72
186
CGPG4700
71643
SENSE

73
187
CGPG475
70242
SENSE

74
188
CGPG478
12325
ANTI-SENSE

75
189
CGPG497
70356
SENSE

76
190
CGPG5283
72034
SENSE

77
191
CGPG5284
72046
SENSE

78
192
CGPG5294
72071
SENSE

79
193
CGPG5308
72116
SENSE

80
194
CGPG5322
72108
SENSE

81
195
CGPG5427
77605
SENSE

82
196
CGPG5595
73978
SENSE

83
197
CGPG5806
73032
SENSE

84
198
CGPG626
11157
SENSE

85
199
CGPG6434
73493
SENSE

86
200
CGPG688
71228
ANTI-SENSE

87
201
CGPG7334
74889
SENSE

88
202
CGPG7339
74854
SENSE

89
203
CGPG7350
74891
SENSE

90
204
CGPG7475
75314
SENSE

91
205
CGPG7493
75340
SENSE

92
206
CGPG7500
75329
SENSE

93
207
CGPG7598
75453
SENSE

94
208
CGPG7600
75477
SENSE

95
209
CGPG7604
75430
SENSE

96
210
CGPG7615
75467
SENSE

97
211
CGPG7630
75457
SENSE

98
212
CGPG7631
75469
SENSE

99
213
CGPG7639
75470
SENSE

100
214
CGPG7644
75435
SENSE

101
215
CGPG7691
77822
SENSE

102
216
CGPG7696
75505
SENSE

103
217
CGPG7703
75589
SENSE

104
218
CGPG7707
75542
SENSE

105
219
CGPG7731
75545
SENSE

106
220
CGPG7734
75581
SENSE

107
221
CGPG7753
75524
SENSE

108
222
CGPG7836
75658
SENSE

109
223
CGPG7867
75738
SENSE

110
224
CGPG8105
77356
SENSE

111
225
CGPG8132
77362
SENSE

112
226
CGPG8137
77363
SENSE

113
227
CGPG8175
77371
SENSE

114
228
CGPG8180
77941
SENSE








































































































































Recombinant DNA
DNA for use in the present invention to improve traits in plants have a nucleotide sequence of SEQ ID NO:1 through SEQ ID NO:114, as well as the homologs of such DNA molecules. A subset of the DNA for gene suppression aspects of the invention includes fragments of the disclosed full polynucleotides consisting of oligonucleotides of 21 or more consecutive nucleotides. Oligonucleotides the larger molecules having a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 114 are useful as probes and primers for detection of the polynucleotides used in the invention. Also useful in this invention are variants of the DNA. Such variants can be naturally occurring, including DNA from homologous genes from the same or a different species, or can be non-natural variants, for example DNA synthesized using chemical synthesis methods, or generated using recombinant DNA techniques. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, a DNA useful in the present invention can have any base sequence that has been changed from the sequences provided herein by substitution in accordance with degeneracy of the genetic code.
Homologs of the genes providing DNA demonstrated as useful in improving traits in model plants disclosed herein will generally have significant identity with the DNA disclosed herein. DNA is substantially identical to a reference DNA if, when the sequences of the polynucleotides are optimally aligned there is at least about 60% nucleotide equivalence over a comparison window. The DNA can also be about 70% equivalence, about 80% equivalence; about 85% equivalence; about 90%; about 95%; or even about 98% or 99% equivalence over a comparison window. A comparison window is at least about 50-100 nucleotides, and/or is the entire length of the polynucleotide provided herein. Optimal alignment of sequences for aligning a comparison window can be conducted by algorithms or by computerized implementations of these algorithms (for example, the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference polynucleotide can be a full-length molecule or a portion of a longer molecule. In one embodiment, the window of comparison for determining polynucleotide identity of protein encoding sequences is the entire coding region.
Proteins useful for imparting enhanced traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins used for generation of transgenic plants having enhanced traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 115 through SEQ ID NO: 228, as well as homologs of such proteins.
Homologs of the trait-improving proteins provided herein generally demonstrate significant sequence identity. Of particular interest are proteins having at least about 50% sequence identity, at least about 70% sequence identity or higher, e.g., at least about 80% sequence identity with an amino acid sequence of SEQ ID NO:115 through SEQ ID NO: 228. Useful proteins also include those with higher identity, e.g., at lease about 90% to at least about 99% identity. Identity of protein homologs is determined by aligning the amino acid sequence of a putative protein homolog with a defined amino acid sequence and by calculating the percentage of identical and conservatively substituted amino acids over the window of comparison. The window of comparison for determining identity can be the entire amino acid sequence disclosed herein, e.g., the full sequence of any of SEQ ID NO: 115 through SEQ ID NO: 228.
The relationship of homologs with amino acid sequences of SEQ ID NO: 229 to SEQ ID NO: 4815 to the proteins with amino acid sequences of SEQ ID NO: to 115 to SEQ ID NO: 228 are found in the listing of Table 16.
Other functional homolog proteins differ in one or more amino acids from those of a trait-improving protein disclosed herein as the result of one or more of the well-known conservative amino acid substitutions, e.g., valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native sequence can be selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the invention includes proteins that differ in one or more amino acids from those of a described protein sequence as the result of deletion or insertion of one or more amino acids in a native sequence.
Genes that are homologous to each other can be grouped into families and included in multiple sequence alignments. Then a consensus sequence for each group can be derived. This analysis enables the derivation of conserved and class- (family) specific residues or motifs that are functionally important. These conserved residues and motifs can be further validated with 3D protein structure if available. The consensus sequence can be used to define the full scope of the invention, e.g., to identify proteins with a homolog relationship. Thus, the present invention contemplates that protein homologs include proteins with an amino acid sequence that has at least 90% identity to such a consensus amino acid sequence sequences.
Promoters
Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens , caulimovirus promoters such as the cauliflower mosaic virus or Figwort mosaic virus promoters. For instance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which disclose versions of the constitutive promoter derived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No. 6,084,089 which discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which discloses light inducible promoters, U.S. Pat. No. 6,140,078 which discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060 which discloses phosphorus deficiency inducible promoters, U.S. Patent Application Publication 2002/0192813A1 which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors, U.S. patent application Ser. No. 09/078,972 which discloses a coixin promoter, U.S. patent application Ser. No. 09/757,089 which discloses a maize chloroplast aldolase promoter, and U.S. patent application Ser. No. 10/739,565 which discloses water-deficit inducible promoters, all of which are incorporated herein by reference. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant polynucleotides of the present invention to provide for expression of desired genes in transgenic plant cells.
Furthermore, the promoters can include multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein can be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, these 5′ enhancing elements are introns. Deemed to be particularly useful as enhancers are the 5′ introns of the rice actin 1 and rice actin 2 genes. Examples of other enhancers that can be used in accordance with the invention include elements from the CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.
In some aspects of the invention, the promoter element in the DNA construct can be capable of causing sufficient expression to result in the production of an effective amount of a polypeptide in water deficit conditions. Such promoters can be identified and isolated from the regulatory region of plant genes that are over expressed in water deficit conditions. Specific water-deficit-inducible promoters for use in this invention are derived from the 5′ regulatory region of genes identified as a heat shock protein 17.5 gene (HSP17.5), an HV A22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea maize . Such water-deficit-inducible promoters are disclosed in U.S. application Ser. No. 10/739,565, incorporated herein by reference.
In some aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al., (1997) Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al., (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy et al., (1996) Plant Mol. Biol. 31(6):1205-1216).
In some aspects of the invention, expression in plant green tissues is desired. Promoters of interest for such uses include those from genes such as SSU (Fischhoff, et al., (1992) Plant Mol. Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).
Gene suppression includes any of the well-known methods for suppressing transcription of a gene or the accumulation of the mRNA corresponding to that gene thereby preventing translation of the transcript into protein. Posttranscriptional gene suppression is mediated by transcription of RNA that forms double-stranded RNA (dsRNA) having homology to a gene targeted for suppression. Suppression can also be achieved by insertion mutations created by transposable elements can also prevent gene function. For example, in many dicot plants, transformation with the T-DNA of Agrobacterium can be readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that can efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest can be identified using the polynucleotides of the present invention. For example, a large population of mutated plants can be screened with polynucleotides encoding the polypeptide of interest to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.
Gene Stacking
The present invention also contemplates that the trait-improving recombinant DNA provided herein can be used in combination with other recombinant DNA to create plants with multiple desired traits or a further enhanced trait. The combinations generated can include multiple copies of any one or more of the recombinant DNA constructs. These stacked combinations can be created by any method, including but not limited to cross breeding of transgenic plants, or multiple genetic transformation.
Transformation Methods
Numerous methods for producing plant cell nuclei with recombinant DNA are known in the art and can be used in the present invention. Two commonly used methods for plant transformation are Agrobacterium -mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. Nos. 5,015,580 (soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soybean); 6,160,208 (corn); 6,399,861 (corn) and 6,153,812 (wheat) and Agrobacterium -mediated transformation is described in U.S. Pat. Nos. 5,159,135 (cotton); 5,824,877 (soybean); 5,463,174 (canola), 5,591,616 (corn); and 6,384,301 (soybean), all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs will include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.
Numerous methods for transforming chromosomes in a plant cell nucleus with recombinant DNA are known in the art and are used in methods of preparing a transgenic plant cell nucleus cell, and plant. Two effective methods for such transformation are Agrobacterium -mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. Nos. 5,015,580 (soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soybean); 6,160,208 (corn); 6,399,861 (corn); 6,153,812 (wheat) and 6,365,807 (rice) and Agrobacterium -mediated transformation is described in U.S. Pat. Nos. 5,159,135 (cotton); 5,824,877 (soybean); 5,463,174 (canola, also known as rapeseed); 5,591,616 (corn); 6,384,301 (soybean), 7,026,528 (wheat) and 6,329,571 (rice), all of which are incorporated herein by reference for enabling the production of transgenic plants. Transformation of plant material is practiced in tissue culture on a nutrient media, i.e. a mixture of nutrients that will allow cells to grow in vitro. Recipient cell targets include, but are not limited to, meristem cells, hypocotyls, calli, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. Callus may be initiated from tissue sources including, but not limited to, immature embryos, hypocotyls, seedling apical meristems, microspores and the like. Cells containing a transgenic nucleus are grown into transgenic plants.
In general it is useful to introduce heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line. In special cases it can be useful to target heterologous DNA insertion in order to achieve site-specific integration, e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function in plants including cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.
Transformation methods of this invention can be practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, calli, hypocotyles, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant can be regenerated is useful as a recipient cell. Callus can be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. patent application Ser. No. 09/757,089, which are incorporated herein by reference.
The seeds of transgenic plants can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plants line for selection of plants having an enhanced trait. In addition to direct transformation of a plant with a recombinant DNA, transgenic plants can be prepared by crossing a first plant having a recombinant DNA with a second plant lacking the DNA. For example, recombinant DNA can be introduced into a first plant line that is amenable to transformation to produce a transgenic plant which can be crossed with a second plant line to introgress the recombinant DNA into the second plant line. A transgenic plant with recombinant DNA providing an enhanced trait, e.g. enhanced yield, can be crossed with transgenic plant line having other recombinant DNA that confers another trait, for example herbicide resistance or pest resistance, to produce progeny plants having recombinant DNA that confers both traits. Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross will segregate such that some of the plants will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA, e.g. marker identification by analysis for recombinant DNA or, in the case where a selectable marker is linked to the recombinant, by application of the selecting agent such as a herbicide for use with a herbicide tolerance marker, or by selection for the enhanced trait. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line
In practice, DNA is introduced into only a small percentage of target cell nuclei. Marker genes are used to provide an efficient system for identification of those cells with nuclei that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes. Some marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells with a nucleus of the invention are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the exogenous DNA in the nucleus. Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptII), hygromycin B (aph IV), spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO) and glyphosate (EPSPS). Examples of such selectable markers are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. See PCT publication WO 99/61129 (herein incorporated by reference) which discloses use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g., an NPTII gene and a GFP gene.
Plant cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, can be cultured in regeneration media and allowed to mature into plants. Developing plantlets can be transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO 2 , and 25-250 microeinsteins m −2 S −1 of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28° C. After regenerating plants have reached the stage of shoot and root development, they can be transferred to a greenhouse for further growth and testing. Plants can be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
Progeny can be recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide. Useful assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
Discovery of Trait-Improving Recombinant DNA
To identify nuclei with recombinant DNA that confer enhanced traits to plants, Arabidopsis thaliana was transformed with a candidate recombinant DNA construct and screened for an enhanced trait.
Arabidopsis thaliana is used a model for genetics and metabolism in plants. A two-step screening process was employed which included two passes of trait characterization to ensure that the trait modification was dependent on expression of the recombinant DNA, but not due to the chromosomal location of the integration of the transgene. Twelve independent transgenic lines for each recombinant DNA construct were established and assayed for the transgene expression levels. Five transgenic lines with high transgene expression levels were used in the first pass screen to evaluate the transgene's function in T2 transgenic plants. Subsequently, three transgenic events, which had been shown to have one or more enhanced traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an enhanced trait. The following Table 3 summarizes the enhanced traits that have been confirmed as provided by a recombinant DNA construct.
In particular, Table 3 reports:
“PEP SEQ ID” which is the amino acid sequence of the protein cognate to the DNA in the recombinant DNA construct corresponding to a protein sequence of a SEQ ID NO. in the Sequence Listing.
“construct_id” is an arbitrary name for the recombinant DNA describe more particularly in Table 1.
“annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (ncbi). More particularly, “gi” is the GenBank ID number for the top BLAST hit.
“description” refers to the description of the top BLAST hit.
“e-value” provides the expectation value for the BLAST hit.
“% id” refers to the percentage of identically matched amino acid residues along the length of the portion of the sequences which is aligned by BLAST between the sequence of interest provided herein and the hit sequence in GenBank.
“traits” identify by two letter codes the confirmed enhancement in a transgenic plant provided by the recombinant DNA. The codes for enhanced traits are:
“CK” which indicates cold tolerance enhancement identified under a cold shock tolerance screen;
“CS” which indicates cold tolerance enhancement identified by a cold germination tolerance screen;
“DS” which indicates drought tolerance enhancement identified by a soil drought stress tolerance screen;
“PEG” which indicates osmotic stress tolerance enhancement identified by a PEG induced osmotic stress tolerance screen;
“HS” which indicates heat stress tolerance enhancement identified by a heat stress tolerance screen;
“SS” which indicates high salinity stress tolerance enhancement identified by a salt stress tolerance screen;
“LN” which indicates nitrogen use efficiency enhancement identified by a limited nitrogen tolerance screen;
“LL” which indicates attenuated shade avoidance response identified by a shade tolerance screen under a low light condition;
“PP” which indicates enhanced growth and development at early stages identified by an early plant growth and development screen;
“SP” which indicates enhanced growth and development at late stages identified by a late plant growth and development screen provided herein.








TABLE 3



PEP




SEQ

Annotation








ID

%




NO
Construct
Id
E-value
Description
traits














115
10177
86
1.00E−100
ref|NP_188965.1|ATERF1/ERF1; DNA
LN









binding/transcription factor/transcriptional





activator [ Arabidopsis thaliana ]

116
12155
85
1.00E−178
ref|NP_565684.1|nucleic acid binding/
CS
SS





transcription factor/zinc ion binding





[ Arabidopsis thaliana ]

117
12233
100
1.00E−144
gb|AAG51950.1|AC015450_11 unknown
LN
SS





protein; 77280-78196 [ Arabidopsis thaliana ]

118
11873
91
0
ref|NP_187289.1|phosphoric diester
LN
PEG





hydrolase/transcription factor [ Arabidopsis





thaliana ]

119
17308
94
0
ref|NP_198771.1|protein binding/
CK





ubiquitin-protein ligase/zinc ion binding





[ Arabidopsis thaliana ]

120
78706
80
2.00E−75
ref|NP_188145.1|SPL5 (SQUAMOSA
PEG





PROMOTER BINDING PROTEIN-LIKE





5); DNA binding/transcription factor





[ Arabidopsis thaliana ]

121
72666
100
1.00E−135
ref|NP_181657.1|DNA binding/
SP





transcription factor [ Arabidopsis thaliana ]





gb|AAC78547.1|

122
17520
74
4.00E−75
gb|ABK28523.1|unknown [ Arabidopsis
CK
HS
PP
PEG





thaliana ]

123
72672
100
8.00E−91
ref|NP_192762.1|transcription factor/zinc
CS





ion binding [ Arabidopsis thaliana ]

124
78977
80
2.00E−83
ref|NP_173506.1|protein binding/
LN





ubiquitin-protein ligase/zinc ion binding





[ Arabidopsis thaliana ]

125
17468
75
1.00E−109
ref|NP_195167.1|DNA binding/
CS





transcription factor [ Arabidopsis thaliana ]

126
75090
70
1.00E−166
ref|NP_194111.1|transcription factor
HS
PP
SS
PEG





[ Arabidopsis thaliana ]

127
72018
85
5.00E−85
ref|NP_179337.1|RHA3A; protein binding/
DS
LL
LN





ubiquitin-protein ligase/zinc ion binding





[ Arabidopsis thaliana ]

128
17524
97
0
ref|NP_200014.1|nucleic acid binding/
CK
LL
PEG





transcription factor/zinc ion binding





[ Arabidopsis thaliana ]

129
72078
74
1.00E−126
ref|NP_190677.1|transcription factor
LL





[ Arabidopsis thaliana ]

130
72977
94
0
ref|NP_850745.1|DNA binding/
CK
LN





transcription factor [ Arabidopsis thaliana ]

131
17445
72
1.00E−100
ref|NP_191608.1|DNA binding/
LN





transcription factor [ Arabidopsis thaliana ]

132
78979
82
1.00E−153
ref|NP_191212.1|ANAC064; transcription
LL
LN





factor [ Arabidopsis thaliana ]

133
18453
69
1.00E−154
ref|NP_174001.1|DNA binding/
CS
HS
PP
SS





transcription factor [ Arabidopsis thaliana ]

134
17913
88
8.00E−69
ref|NP_196812.1|WRKY75; transcription
CK
HS





factor [ Arabidopsis thaliana ]

135
18210
78
2.00E−82
ref|NP_563624.1|DNA binding/
PEG





transcription factor [ Arabidopsis thaliana ]

136
78710
93
1.00E−154
gb|AAF79495.1|AC002328_3F20N2.8
HS
LN





[ Arabidopsis thaliana ]

137
73606
83
1.00E−108
ref|NP_196295.1|nucleic acid binding/
CS
HS
SS





transcription factor [ Arabidopsis thaliana ]

138
18446
100
1.00E−166
ref|NP_176212.1|transcription factor
SS





[ Arabidopsis thaliana ]

139
75011
78
1.00E−134
ref|NP_195651.1|WRKY13; transcription
PEG





factor [ Arabidopsis thaliana ]

140
18507
68
1.00E−119
ref|NP_191581.1|ATL4; protein binding/
CK
SS





ubiquitin-protein ligase/zinc ion binding





[ Arabidopsis thaliana ]

141
18388
93
1.00E−133
ref|NP_190147.1|DNA binding/
SS





transcription factor [ Arabidopsis thaliana ]

142
18540
82
1.00E−110
ref|NP_193836.1|DNA binding/
PP
PEG





transcription factor [ Arabidopsis thaliana ]

143
19653
83
3.00E−72
gb|ABB02372.1|AP2/EREBP transcription
CS
LN





factor [ Arabidopsis thaliana ]

144
18411
80
3.00E−93
gb|AAD20668.1|ethylene reponse factor-
CS
HS





like AP2 domain transcription factor





[ Arabidopsis thaliana ]

145
70737
100
0
ref|NP_564265.1|unknown protein
CS
HS
SS





[ Arabidopsis thaliana ]

146
18315
72
3.00E−90
ref|NP_196054.1|C2H2; nucleic acid
SS
PEG





binding/transcription factor/zinc ion





binding [ Arabidopsis thaliana ]

147
75080
94
1.00E−117
ref|NP_179033.1|ANR1; DNA binding/
HS
PEG





transcription factor [ Arabidopsis thaliana ]

148
19191
71
6.00E−68
gb|AAK76521.1|putative Dof zinc finger
SS





protein [ Arabidopsis thaliana ]

149
18431
90
0
ref|NP_192961.1|nucleic acid binding/
LN





transcription factor/zinc ion binding





[ Arabidopsis thaliana ]

150
18433
89
1.00E−163
ref|NP_195443.1|MYB73; DNA binding/
SP





transcription factor [ Arabidopsis thaliana ]

151
18434
72
1.00E−122
ref|NP_195758.1|transcription factor
HS





[ Arabidopsis thaliana ]

152
19536
93
0
ref|NP_568099.1/MYB3R-5; DNA binding/
PEG





transcription factor [ Arabidopsis thaliana ]

153
76577
93
8.00E−84
ref|NP_180991.1|transcription factor
PEG





[ Arabidopsis thaliana ]

154
11113
92
0
ref|NP_565621.1|PEX10; protein binding/
CS





ubiquitin-protein ligase/zinc ion binding





[ Arabidopsis thaliana ]

155
19614
100
1.00E−84
ref|NP_566925.1|DNA binding/
PEG





transcription factor [ Arabidopsis thaliana ]

156
18204
85
0
ref|NP_001031255.1|unknown protein
CK





[ Arabidopsis thaliana ]

157
73614
96
0
ref|NP_186995.1|RGL2 (RGA-LIKE 2);
CS
HS





transcription factor [ Arabidopsis thaliana ]

158
70450
76
1.00E−138
ref|NP_187687.1|DNA binding/
CS
HS





transcription factor [ Arabidopsis thaliana ]

159
70618
94
0
ref|NP_188000.1|transcription factor
HS
SS





[ Arabidopsis thaliana ]

160
70458
95
0
ref|NP_172779.1|unknown protein
LL





[ Arabidopsis thaliana ]

161
71306
81
0
ref|NP_195480.1|SHR (SHORT ROOT);
CS
HS
SP
PEG





transcription factor [ Arabidopsis thaliana ]

162
70451
92
1.00E−141
ref|NP_195810.2|WRKY62; transcription
SS





factor [ Arabidopsis thaliana ]

163
70453
71
1.00E−154
ref|NP_201032.2|DNA binding
CS





[ Arabidopsis thaliana ]

164
71311
89
0
ref|NP_176498.1|transcription factor
CS
LN





[ Arabidopsis thaliana ]

165
76533
91
0
gb|ABK28765.1|unknown [ Arabidopsis
HS





thaliana ]

166
70955
87
1.00E−178
gb|AAX21351.1|phantastica transcription
SP





factor a [ Glycine max ]

167
77708
57
1.00E−64
ref|NP_200279.1|DNA binding/
DS





transcription factor [ Arabidopsis thaliana ]

168
70954
61
9.00E−72
emb|CAB77055.1|putative TFIIIA (or
LN
PEG





kruppel)-like zinc finger protein [ Medicago





sativa subsp. × varia]

169
19742
86
1.00E−156
gb|AAK84883.1|AF402602_1NAC domain
CS





protein NAC1 [ Phaseolus vulgaris ]

170
70960
53
1.00E−72
ref|NP_565183.1|transcription factor/zinc
PEG





ion binding [ Arabidopsis thaliana ]

171
19940
94
0
gb|ABH02845.1|MYB transcription factor
PEG





MYB93 [ Glycine max ]

172
70921
88
1.00E−156
gb|AAX85979.1|NAC2 protein [ Glycine
CS
CK





max ]

173
70953
83
1.00E−121
gb|ABI97244.1|PHD5 [ Glycine max ]
DS
PP
SS
PEG

174
70978
99
1.00E−134
emb|CAI47596.1|MADS transcription factor
HS
PEG





[ Glycine max ]

175
70940
47
1.00E−97
ref|NP_191362.2|protein binding/
SS





ubiquitin-protein ligase/zinc ion binding





[ Arabidopsis thaliana ]

176
70977
57
8.00E−76
ref|NP_567245.1|DNA binding/
HS





transcription factor [ Arabidopsis thaliana ]

177
70993
92
1.00E−105
gb|AAX13302.1|MADS box protein AP3-
CS
SS





like [ Lotus corniculatus var. japonicus]

178
19762
98
2.00E−36
gb|ABH02867.1|MYB transcription factor
HS
PP





MYB142 [ Glycine max ]

179
70938
47
2.00E−63
dbj|BAB16432.1|WRKY transcription
CS
HS
PP
PEG





factor NtEIG-D48 [ Nicotiana tabacum ]

180
78653
100
1.00E−114
ref|NP_199999.1|transcription factor
LL
LN





[ Arabidopsis thaliana ]

181
73216
99
0
ref|NP_189228.1|transcription factor
LL





[ Arabidopsis thaliana ]

182
71139
91
1.00E−152
ref|NP_187669.1|DNA binding/
LN





transcription factor [ Arabidopsis thaliana ]

183
74227
74
1.00E−174
ref|NP_187615.2|transcription factor
LL





[ Arabidopsis thaliana ]

184
70678
91
1.00E−142
ref|NP_173195.2|DNA binding/
LN
LL





transcription factor [ Arabidopsis thaliana ]

185
70769
90
1.00E−148
ref|NP_194286.1|DNA binding/
LL





transcription factor [ Arabidopsis thaliana ]

186
71643
83
1.00E−141
gb|ABH02851.1|MYB transcription factor
LL
LN





MYB109 [ Glycine max ]

187
70242
93
0
ref|NP_565162.1|calmodulin binding/
DS





transcription factor [ Arabidopsis thaliana ]

188
12325
94
1.00E−164
ref|NP_191684.1|DNA binding/
LN





transcription factor [ Arabidopsis thaliana ]

189
70356
95
1.00E−114
gb|AAX13306.1|MADS box protein AGL11
DS





[ Lotus corniculatus var. japonicus]

190
72034
72
3.00E−90
gb|AAX69070.1|MADS box protein M8
LL





[ Pisum sativum ]

191
72046
57
1.00E−64
dbj|BAD15085.1|CCAAT-box binding
CS
PP
SS





factor HAP5 homolog [ Daucus carota ]

192
72071
76
1.00E−79
sp|P32294|AX22B_PHAAUAuxin-induced
PEG





protein 22B (Indole-3-acetic acid-induced





protein ARG4) dbj|BAA03309.1|ORF





[ Vigna radiata ]

193
72116
93
1.00E−123
dbj|BAB86892.1|syringolide-induced
HS
LN
SS





protein 1-3-1A [ Glycine max ]

194
72108
82
1.00E−87
gb|ABK58464.1|LIM domain protein
LL





GLIM1a [ Populus tremula × Populus alba ]

195
77605
99
0
gb|ABE65879.1|zinc finger family protein
CK
HS
LL
PEG
PP





[ Arabidopsis thaliana ]

196
73978
90
0
ref|NP_179919.1|CLF (CURLY LEAF);
SS
PEG





transcription factor [ Arabidopsis thaliana ]

197
73032
69
1.00E−115
gb|ABI34669.1|bZIP transcription factor
LL





bZIP131 [ Glycine max ]

198
11157
96
0
ref|NP_177279.1|AJH2 [ Arabidopsis
LN





thaliana ]

199
73493
97
0
ref|NP_010736.1|Ada2p [ Saccharomyces
PEG





cerevisiae ]

200
71228
93
1.00E−135
ref|NP_565948.1|EEL (ENHANCED EM
CK





LEVEL); DNA binding/transcription





factor [ Arabidopsis thaliana ]

201
74889
64
1.00E−118
gb|ABE82375.1|Transcription factor
CS





E2F/dimerisation partner (TDP) [ Medicago





truncatula ]

202
74854
75
1.00E−115
gb|ABH02826.1|MYB transcription factor
LN





MYB55 [ Glycine max ]

203
74891
88
1.00E−67
gb|AAL31068.1|AC090120_14putative c-
CS
DS
LN





myc binding protein [ Oryza sativa ]

204
75314
51
5.00E−71
ref|NP_181828.1|ANAC042; transcription
DS
LN





factor [ Arabidopsis thaliana ]

205
75340
66
3.00E−88
emb|CAA63222.1|homeobox-leucine zipper
LL





protein [ Glycine max ]

206
75329
91
1.00E−62
ref|NP_001062531.1|Os08g0564500 [ Oryza
PEG





sativa (japonica cultivar-group)]

207
75453
81
1.00E−140
gb|ABH02828.1|MYB transcription factor
HS
LN





MYB57 [ Glycine max ]

208
75477
48
5.00E−38
gb|ABC69353.1|ethylene-responsive
LL
PEG





element-binding protein [ Medicago





truncatula ]

209
75430
69
9.00E−30
emb|CAC28528.1|GATA-1 zinc finger
LL
LN
PEG





protein [ Nicotiana tabacum ]

210
75467
43
3.00E−30
gb|AAM61659.1|unknown [ Arabidopsis
CS
HS





thaliana ]

211
75457
72
1.00E−89
gb|AAX69065.1|MADS box protein M2
PP
SP





[ Pisum sativum ]

212
75469
91
1.00E−143
gb|ABI34650.1|bZIP transcription factor
SP





bZIP68 [ Glycine max ]

213
75470
74
2.00E−44
gb|AAP13348.1|transcription factor GT-3b
HS
PP





[ Arabidopsis thaliana ]

214
75435
71
1.00E−28
ref|NP_566164.1|PTF1 (PLASTID
DS





TRANSCRIPTION FACTOR 1);





transcription factor [ Arabidopsis thaliana ]

215
77822
61
2.00E−75
gb|AAN05420.1|putative RING protein
DS
LL





[ Populus × canescens ]

216
75505
49
1.00E−172
gb|ABI30334.1|Cys-3-His zinc finger
PP





protein [ Capsicum annuum ]

217
75589
56
1.00E−115
gb|ABE81808.1|Homeodomain-related
LN





[ Medicago truncatula ]

218
75542
69
9.00E−83
gb|ABE78575.1|TCP transcription factor
LN
SP





[ Medicago truncatula ]

219
75545
42
2.00E−36
gb|AAQ56115.1|transcription-factor-like
SS





protein [ Boechera drummondii ]

220
75581
44
1.00E−89
ref|NP_189124.1|PRT1 (PROTEOLYSIS
LL





1); ubiquitin-protein ligase [ Arabidopsis





thaliana ]

221
75524
50
1.00E−106
ref|NP_176980.1|nucleic acid binding/
LN





transcription factor/zinc ion binding





[ Arabidopsis thaliana ]

222
75658
49
6.00E−29
gb|AAV85852.1|AT-rich element binding
LL





factor 2 [ Pisum sativum ]

223
75738
67
6.00E−68
gb|AAM33103.2|TAGL12 transcription
LL
SP





factor [ Lycopersicon esculentum ]

224
77356
94
1.00E−119
ref|NP_564547.1|RTV1; DNA binding/
LL





transcription factor [ Arabidopsis thaliana ]

225
77362
100
3.00E−73
ref|NP_193892.1|LOL2 (LSD ONE LIKE
DS





2); transcription factor [ Arabidopsis





thaliana ]

226
77363
67
1.00E−47
ref|NP_194747.1|transcription factor/
LN





transcription regulator [ Arabidopsis





thaliana ]

227
77371
95
1.00E−127
ref|NP_199178.1|DNA binding/
CS





transcription factor [ Arabidopsis thaliana ]

228
77941
90
1.00E−140
dbj|BAB09311.1|tumor-related protein-like
DS
SS





[ Arabidopsis thaliana ]























































































































































































































































































Trait Enhancement Screens
DS-Enhancement of drought tolerance identified by a soil drought stress tolerance screen: Drought or water deficit conditions impose mainly osmotic stress on plants. Plants are particularly vulnerable to drought during the flowering stage. The drought condition in the screening process disclosed in Example 1B started from the flowering time and was sustained to the end of harvesting. The present invention provides recombinant DNA that can improve the plant survival rate under such sustained drought condition. Exemplary recombinant DNA for conferring such drought tolerance are identified as such in Table 3. Such recombinant DNA can be used in generating transgenic plants that are tolerant to the drought condition imposed during flowering time and in other stages of the plant life cycle. As demonstrated from the model plant screen, in some embodiments of transgenic plants with trait-improving recombinant DNA grown under such sustained drought condition can also have increased total seed weight per plant in addition to the increased survival rate within a transgenic population, providing a higher yield potential as compared to control plants.
PEG-Enhancement of drought tolerance identified by PEG induced osmotic stress tolerance screen: Various drought levels can be artificially induced by using various concentrations of polyethylene glycol (PEG) to produce different osmotic potentials (Pilon-Smits e.g., (1995) Plant Physiol. 107:125-130). Several physiological characteristics have been reported as being reliable indications for selection of plants possessing drought tolerance. These characteristics include the rate of seed germination and seedling growth. The traits can be assayed relatively easily by measuring the growth rate of seedling in PEG solution. Thus, a PEG-induced osmotic stress tolerance screen is a useful surrogate for drought tolerance screen. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the PEG-induced osmotic stress tolerance screen can survive better drought conditions providing a higher yield potential as compared to control plants.
SS-Enhancement of drought tolerance identified by high salinity stress tolerance screen: Three different factors are responsible for salt damages: (1) osmotic effects, (2) disturbances in the mineralization process, and (3) toxic effects caused by the salt ions, e.g., inactivation of enzymes. While the first factor of salt stress results in the wilting of the plants that is similar to drought effect, the ionic aspect of salt stress is clearly distinct from drought. The present invention provides genes that help plants maintain biomass, root growth, and/or plant development in high salinity conditions, which are identified as such in Table 3. Since osmotic effect is one of the major components of salt stress, which is common to the drought stress, trait-improving recombinant DNA identified in a high salinity stress tolerance screen can also provide transgenic crops with enhanced drought tolerance. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a high salinity stress tolerance screen can survive better drought conditions and/or high salinity conditions providing a higher yield potential as compared to control plants.
HS-Enhancement of drought tolerance identified by heat stress tolerance screen: Heat and drought stress often occur simultaneously, limiting plant growth. Heat stress can cause the reduction in photosynthesis rate, inhibition of leaf growth and osmotic potential in plants. Thus, genes identified by the present invention as heat stress tolerance conferring genes can also impart enhanced drought tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a heat stress tolerance screen can survive better heat stress conditions and/or drought conditions providing a higher yield potential as compared to control plants.
CK and CS-Enhancement of tolerance to cold stress: Low temperature can immediately result in mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential. In the present invention, two screening conditions, i.e., cold shock tolerance screen (CK) and cold germination tolerance screen (CS), were set up to look for transgenic plants that display visual growth advantage at lower temperature. In cold germination tolerance screen, the transgenic Arabidopsis plants were exposed to a constant temperature of 8° C. from planting until day 28 post plating. The trait-improving recombinant DNA identified by such screen are particular useful for the production of transgenic plant that can germinate more robustly in a cold temperature as compared to the wild type plants. In cold shock tolerance screen, the transgenic plants were first grown under the normal growth temperature of 22° C. until day 8 post plating, and subsequently were placed under 8° C. until day 28 post plating. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a cold shock stress tolerance screen and/or a cold germination stress tolerance screen can survive better cold conditions providing a higher yield potential as compared to control plants.
Enhancement of tolerance to multiple stresses: Different kinds of stresses often lead to identical or similar reaction in the plants. Genes that are activated or inactivated as a reaction to stress can either act directly in a way the genetic product reduces a specific stress, or they can act indirectly by activating other specific stress genes. By manipulating the activity of such regulatory genes, i.e., multiple stress tolerance genes, the plant can be enabled to react to different kinds of stresses. For examples, PEP SEQ ID NO: 116 can be used to enhance both salt stress tolerance and cold stress tolerance in plants. Of particular interest, plants transformed with PEP SEQ ID NO: 133 can resist salt stress and cold stress. Plants transformed with PEP SEQ ID NO: 133 can also improve growth in early stage and under osmotic stress. In addition to these multiple stress tolerance genes, the stress tolerance conferring genes provided by the present invention can be used in combinations to generate transgenic plants that can resist multiple stress conditions.
PP-Enhancement of early plant growth and development: It has been known in the art that to minimize the impact of disease on crop profitability, it is important to start the season with healthy and vigorous plants. This means avoiding seed and seedling diseases, leading to increased nutrient uptake and increased yield potential. Traditionally early planting and applying fertilizer are the methods used for promoting early seedling vigor. In early development stage, plant embryos establish only the basic root-shoot axis, a cotyledon storage organ(s), and stem cell populations, called the root and shoot apical meristems that continuously generate new organs throughout post-embryonic development. “Early growth and development” used herein encompasses the stages of seed imbibition through the early vegetative phase. The present invention provides genes that are useful to produce transgenic plants that have advantages in one or more processes including, but not limited to, germination, seedling vigor, root growth and root morphology under non-stressed conditions. The transgenic plants starting from a more robust seedling are less susceptible to the fungal and bacterial pathogens that attach germinating seeds and seedling. Furthermore, seedlings with advantage in root growth are more resistant to drought stress due to extensive and deeper root architecture. Therefore, it can be recognized by those skilled in the art that genes conferring the growth advantage in early stages to plants can also be used to generate transgenic plants that are more resistant to various stress conditions due to enhanced early plant development. The present invention provides such exemplary recombinant DNA that confer both the stress tolerance and growth advantages to plants, identified as such in Table 3, e.g., PEP SEQ ID NO: 126 which can improve the plant early growth and development, and impart salt tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the early plant development screen can grow better under non-stress conditions and/or stress conditions providing a higher yield potential as compared to control plants.
SP-Enhancement of late plant growth and development: “Late growth and development” used herein encompasses the stages of leaf development, flower production, and seed maturity. In certain embodiments, transgenic plants produced using genes that confer growth advantages to plants provided by the present invention, identified as such in Table 3, exhibit at least one phenotypic characteristics including, but not limited to, increased rosette radius, increased rosette dry weight, seed dry weight, silique dry weight, and silique length. On one hand, the rosette radius and rosette dry weight are used as the indexes of photosynthesis capacity, and thereby plant source strength and yield potential of a plant. On the other hand, the seed dry weight, silique dry weight and silique length are used as the indexes for plant sink strength, which are considered as the direct determinants of yield. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen can grow better and/or have enhanced development during leaf development and seed maturation providing a higher yield potential as compared to control plants.
LL-Enhancement of tolerance to shade stress identified in a low light screen: The effects of light on plant development are especially prominent at the seedling stage. Under normal light conditions with unobstructed direct light, a plant seeding develops according to a characteristic photomorphogenic pattern, in which plants have open and expanded cotyledons and short hypocotyls. Then the plant's energy is devoted to cotyledon and leaf development while longitudinal extension growth is minimized. Under low light condition where light quality and intensity are reduced by shading, obstruction or high population density, a seedling displays a shade-avoidance pattern, in which the seedling displays a reduced cotyledon expansion, and hypocotyls extension is greatly increased. As the result, a plant under low light condition increases significantly its stem length at the expanse of leaf, seed or fruit and storage organ development, thereby adversely affecting of yield. The present invention provides recombinant DNA that enable plants to have an attenuated shade avoidance response so that the source of plant can be contributed to reproductive growth efficiently, resulting higher yield as compared to the wild type plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a shade stress tolerance screen can have attenuated shade response under shade conditions providing a higher yield potential as compared to control plants. The transgenic plants generated by the present invention can be suitable for a higher density planting, thereby resulting increased yield per unit area.
LN-Enhancement of Tolerance to Low Nitrogen Availability Stress
Nitrogen is a key factor in plant growth and crop yield. The metabolism, growth and development of plants are profoundly affected by their nitrogen supply. Restricted nitrogen supply alters shoot to root ratio, root development, activity of enzymes of primary metabolism and the rate of senescence (death) of older leaves. All field crops have a fundamental dependence on inorganic nitrogenous fertilizer. Since fertilizer is rapidly depleted from most soil types, it must be supplied to growing crops two or three times during the growing season. Enhanced nitrogen use efficiency by plants should enable crops cultivated under low nitrogen availability stress condition resulted from low fertilizer input or poor soil quality.
This invention demonstrates that the transgenic plants generated using the recombinant nucleotides, which confer enhanced nitrogen use efficiency, identified as such in Table 3, exhibit one or more desirable traits including, but not limited to, increased seedling weight, greener leaves, increased number of rosette leaves, increased or decreased root length. One skilled in the art can recognize that the transgenic plants provided by the present invention with enhanced nitrogen use efficiency can also have altered amino acid or protein compositions, increased yield and/or better seed quality. The transgenic plants of the present invention can be productively cultivated under low nitrogen growth conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer inputs, which would cause the growth of wild type plants to cease or to be so diminished as to make the wild type plants practically useless. The transgenic plants also can be advantageously used to achieve earlier maturing, faster growing, and/or higher yielding crops and/or produce more nutritious foods and animal feedstocks when cultivated using nitrogen non-limiting growth conditions.
Stacked Traits: The present invention also encompasses transgenic plants with stacked engineered traits, e.g., a crop having an enhanced phenotype resulting from expression of a trait-improving recombinant DNA, in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, for example a RoundUp Ready® trait, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides. To illustrate that the production of transgenic plants with herbicide resistance is a capability of those of ordinary skill in the art, reference is made to U.S. patent application publications 2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549 and 6,376,754, all of which are incorporated herein by reference. To illustrate that the production of transgenic plants with pest resistance is a capability of those of ordinary skill in the art, reference is made to U.S. Pat. Nos. 5,250,515 and 5,880,275 which disclose plants expressing an endotoxin of Bacillus thuringiensis bacteria, to U.S. Pat. No. 6,506,599 which discloses control of invertebrates which feed on transgenic plants which express dsRNA for suppressing a target gene in the invertebrate, to U.S. Pat. No. 5,986,175 which discloses the control of viral pests by transgenic plants which express viral replicase, and to U.S. Patent Application Publication 2003/0150017 A1 which discloses control of pests by a transgenic plant which express a dsRNA targeted to suppressing a gene in the pest, all of which are incorporated herein by reference.
Once one recombinant DNA has been identified as conferring an enhanced trait of interest in transgenic Arabidopsis plants, several methods are available for using the sequence of that recombinant DNA and knowledge about the protein it encodes to identify homologs of that sequence from the same plant or different plant species or other organisms, e.g., bacteria and yeast. Thus, in one aspect, the invention provides methods for identifying a homologous gene with a DNA sequence homologous to any of SEQ ID NO: 1 through SEQ ID NO: 114, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO: 115 through SEQ ID NO: 228. In another aspect, the present invention provides the protein sequences of identified homologs for a sequence listed as SEQ ID NO: 229 through SEQ ID NO: 4815. In yet another aspect, the present invention also includes linking or associating one or more desired traits, or gene function with a homolog sequence provided herein.
The trait-improving recombinant DNA and methods of using such trait-improving recombinant DNA for generating transgenic plants with enhanced traits provided by the present invention are not limited to any particular plant species. Indeed, the plants according to the present invention can be of any plant species, i.e., can be monocotyledonous or dicotyledonous. In one embodiment, they will be agricultural useful plants, i.e., plants cultivated by man for purposes of food production or technical, particularly industrial applications. Of particular interest in the present invention are corn and soybean plants. The recombinant DNA constructs optimized for soybean transformation and recombinant DNA constructs optimized for corn transformation are provided by the present invention. Other plants of interest in the present invention for production of transgenic plants having enhanced traits include, without limitation, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
In certain embodiments, the present invention contemplates to use an orthologous gene in generating the transgenic plants with similarly enhanced traits as the transgenic Arabidopsis counterpart. Enhanced physiological properties in transgenic plants of the present invention can be confirmed in responses to stress conditions, for example in assays using imposed stress conditions to detect enhanced responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions. Biomass measures can be made on greenhouse or field grown plants and can include such measurements as plant height, stem diameter, root and shoot dry weights, and, for corn plants, ear length and diameter.
Trait data on morphological changes can be collected by visual observation during the process of plant regeneration as well as in regenerated plants transferred to soil. Such trait data includes characteristics such as normal plants, bushy plants, taller plants, thicker stalks, narrow leaves, striped leaves, knotted phenotype, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other enhanced traits can be identified by measurements taken under field conditions, such as days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance. In addition, trait characteristics of harvested grain can be confirmed, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.
To confirm hybrid yield in transgenic corn plants expressing genes of the present invention, it can be desirable to test hybrids over multiple years at multiple locations in a geographical location where maize is conventionally grown, e.g., in Iowa, Illinois or other locations in the midwestern United States, under “normal” field conditions as well as under stress conditions, e.g., under drought or population density stress.
Transgenic plants can be used to provide plant parts according to the invention for regeneration or tissue culture of cells or tissues containing the constructs described herein. Plant parts for these purposes can include leaves, stems, roots, flowers, tissues, epicotyl, meristems, hypocotyls, cotyledons, pollen, ovaries, cells and protoplasts, or any other portion of the plant which can be used to regenerate additional transgenic plants, cells, protoplasts or tissue culture. Seeds of transgenic plants are provided by this invention can be used to propagate more plants containing the trait-improving recombinant DNA constructs of this invention. These descendants are intended to be included in the scope of this invention if they contain a trait-improving recombinant DNA construct of this invention, whether or not these plants are selfed or crossed with different varieties of plants.
The various aspects of the invention are illustrated by means of the following examples which are in no way intended to limit the full breath and scope of claims.
EXAMPLES
Example 1
Identification of Recombinant DNA that Confers Enhanced Trait(s) to Plants
A. Plant Expression Constructs for Arabidopsis Transformation
Each gene of interest was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. Transformation vectors were prepared to constitutively transcribe DNA in either sense orientation (for enhanced protein expression) or anti-sense orientation (for endogenous gene suppression) under the control of an enhanced Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No. 5,359,142) directly or indirectly (Moore, e.g., PNAS 95:376-381, 1998; Guyer, e.g., Genetics 149: 633-639, 1998; International patent application NO. PCT/EP98/07577). The transformation vectors also contain a bar gene as a selectable marker for resistance to glufosinate herbicide. The transformation of Arabidopsis plants was carried out using the vacuum infiltration method known in the art (Bethtold, e.g., Methods Mol. Biol. 82:259-66, 1998). Seeds harvested from the plants, named as T1 seeds, were subsequently grown in a glufosinate-containing selective medium to select for plants which were actually transformed and which produced T2 transgenic seed.
B. Soil Drought Tolerance Screen
This example describes a soil drought tolerance screen to identify Arabidopsis plants transformed with recombinant DNA that wilt less rapidly and/or produce higher seed yield when grown in soil under drought conditions
T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts® Company, USA). Humidity domes were added to each flat and flats were assigned locations and placed in climate-controlled growth chambers. Plants were grown under a temperature regime of 22° C. at day and 20° C. at night, with a photoperiod of 16 hours and average light intensity of 170 μmol/m 2 /s. After the first true leaves appeared, humidity domes were removed. The plants were sprayed with glufosinate herbicide and put back in the growth chamber for 3 additional days. Flats were watered for 1 hour the week following the herbicide treatment. Watering was continued every seven days until the flower bud primordia became apparent, at which time plants were watered for the last time.
To identify drought tolerant plants, plants were evaluated for wilting response and seed yield. Beginning ten days after the last watering, plants were examined daily until 4 plants/line had wilted. In the next six days, plants were monitored for wilting response. Five drought scores were assigned according to the visual inspection of the phenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severe wilting, and 5 for dead. A score of 3 or higher was considered as wilted.
At the end of this assay, seed yield measured as seed weight per plant under the drought condition was characterized for the transgenic plants and their controls and analyzed as a quantitative response according to example 1M.
Two approaches were used for statistical analysis on the wilting response. First, the risk score was analyzed for wilting phenotype and treated as a qualitative response according to the example 1L. Alternatively, the survival analysis was carried out in which the proportions of wilted and non-wilted transgenic and control plants were compared over each of the six days under scoring and an overall log rank test was performed to compare the two survival curves using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). A list of recombinant DNA constructs which improve drought tolerance in transgenic plants is illustrated in Table 4
TABLE 4 Time to wilting PEP Drought score Seed yield Risk SEQ Construct Delta Delta score ID NO ID Orientation mean P-value mean P-value mean P-value 187 70242 SENSE 0.034 0.539 0.783 0.029 −0.047 1.000 189 70356 SENSE −0.170 0.013 0.193 0.053 −0.170 1.000 176 70977 SENSE 0.010 0.815 0.652 0.015 0.115 1.000 214 75435 SENSE 0.312 0.021 −2.969 0.004 0.248 1.000 225 77362 SENSE 0.076 0.310 0.420 0.068 0.060 0.920 167 77708 SENSE 0.172 0.072 0.342 0.017 −0.038 0.064 129 72078 SENSE 1.794 0.006 / / / /
Transgenic plants including recombinant DNA expressing protein as set forth in SEQ ID NO: 127, 173, 203, 204, 215, or 228 showed enhanced drought tolerance by the second criterial as illustrated in Example 1L.

C. Heat Stress Tolerance Screen
Under high temperatures, Arabidopsis seedlings become chlorotic and root growth is inhibited. This example sets forth the heat stress tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are more resistant to heat stress based on primarily their seedling weight and root growth under high temperature.
T2 seeds were plated on ½×MS salts, 1% phytagel, with 10 μg/ml BASTA (7 per plate with 2 control seeds; 9 seeds total per plate). Plates were placed at 4° C. for 3 days to stratify seeds. Plates were then incubated at room temperature for 3 hours and then held vertically for 11 additional days at temperature of 34° C. at day and 20° C. at night. Photoperiod was 16 h. Average light intensity was ˜140 μmol/m 2 /s. After 14 days of growth, plants were scored for glufosinate resistance, root length, final growth stage, visual color, and seedling fresh weight. A photograph of the whole plate was taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final grow stage at day 14 was scored as success if 50% of the plants had reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, e.g., (2001) The Plant Cell 13, 1499-1510). The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve heat tolerance in transgenic plants illustrated in Table 5.
TABLE 5 Seedling PEP Root length Growth stage weight SEQ ID Construct at day 14 at day 14 at day 14 NO ID Orientation Delta P-value Risk P-value Delta P-value 126 75090 SENSE 0.094 0.410 0.839 0.296 1.000 0.039 133 18453 SENSE 0.253 0.045 0.233 0.359 0.884 0.009 136 78710 SENSE 0.073 0.574 0.139 0.681 0.606 0.008 137 73606 SENSE 0.382 0.058 1.064 0.210 1.237 0.014 145 70737 SENSE −0.007 0.935 0.185 0.048 0.838 0.019 147 75080 SENSE 0.306 0.171 1.172 0.242 1.140 0.021 151 18434 SENSE 0.095 0.385 −0.029 / / 0.030 157 73614 SENSE −0.051 0.238 −0.178 0.377 0.719 0.020 159 70618 SENSE 0.001 0.997 1.812 0.163 0.975 0.030 161 71306 SENSE 0.379 0.004 0.145 0.426 1.542 0.004 165 76533 SENSE 0.090 0.248 0.103 0.182 0.531 0.000 174 70978 SENSE 0.472 0.238 1.308 0.105 1.664 0.026 176 70977 SENSE 0.337 0.015 0.999 0.304 1.193 0.004 178 19762 SENSE 0.042 0.614 −0.259 0.005 0.714 0.024 179 70938 SENSE 0.266 0.094 0.449 0.360 1.024 0.037 195 77605 SENSE 0.167 0.087 0.648 0.392 1.254 0.012 207 75453 SENSE −0.352 0.026 0.517 0.310 0.585 0.013 210 75467 SENSE 0.120 0.438 −0.134 0.069 1.029 0.037 213 75470 SENSE 0.225 0.143 −0.030 0.600 1.135 0.017 158 70450 SENSE 0.354 0.023 0.878 0.006 1.161 0.008
Transgenic plants comprising recombinant DNA expressing protein as set forth in SEQ ID NO: 122, 134, 144 and 193 showed enhanced heat stress tolerance by the second criterial as illustrated in Example 1L and 1M.

D. Salt Stress Tolerance Screen
This example sets forth the high salinity stress screen to identify Arabidopsis plants transformed with the gene of interest that are tolerant to high levels of salt based on their rate of development, root growth and chlorophyll accumulation under high salt conditions.
T2 seeds were plated on glufosinate selection plates containing 90 mM NaCl and grown under standard light and temperature conditions. All seedlings used in the embodiments were grown at a temperature of 22° C. at day and 20° C. at night, a 16-hour photoperiod, an average light intensity of approximately 120 umol/m 2 . On day 11, plants were measured for primary root length. After 3 more days of growth (day 14), plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was also taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success if 50% of the plants reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, D. C., et al., (2001), The Plant Cell 13, 1499/1510). The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve high salinity tolerance in transgenic plants illustrated in Table 6.
TABLE 6 Root length Root length Growth stage Seedling weight PEP at day 11 at day 14 at day 14 at day 14 SEQ Construct Delta Delta Delta Delta ID ID mean P-value mean P-value mean P-value mean P-value 116 12155 0.207 0.258 0.297 0.020 0.691 0.256 0.622 0.012 141 18388 0.415 0.011 0.475 0.009 0.440 0.127 0.385 0.214 138 18446 0.120 0.385 0.120 0.097 / / 0.490 0.042 133 18453 0.265 0.089 0.288 0.060 0.364 0.336 0.356 0.043 148 19191 0.024 0.383 0.104 0.019 0.210 0.466 −0.197 0.034 162 70451 0.185 0.286 0.267 0.195 0.286 0.429 0.713 0.029 159 70618 0.504 0.017 0.535 0.046 0.358 0.403 1.101 0.010 145 70737 0.141 0.157 0.216 0.038 0.983 0.236 0.323 0.208 175 70940 0.668 0.007 0.672 0.004 3.597 0.012 1.201 0.001 173 70953 0.254 0.054 0.297 0.052 1.040 0.413 0.787 0.026 191 72046 0.236 0.010 0.168 0.000 0.502 0.184 0.215 0.177 137 73606 0.399 0.035 0.386 0.041 0.251 0.332 0.284 0.612 196 73978 0.413 0.076 0.327 0.016 0.269 0.423 0.451 0.073 126 75090 0.466 0.024 0.507 0.012 1.340 0.154 1.092 0.036 219 75545 −0.274 0.077 −0.105 0.314 0.197 0.320 0.468 0.018 228 77941 0.341 0.013 0.336 0.041 1.155 0.266 0.574 0.149
Transgenic plants comprising recombinant DNA expressing protein as set forth in SEQ ID NO: 117, 140, 146, 177, or 193 showed enhanced salt stress tolerance by the second criterial as illustrated in Example 1L and 1M.

E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen
There are numerous factors, which can influence seed germination and subsequent seedling growth, one being the availability of water. Genes, which can directly affect the success rate of germination and early seedling growth, are potentially useful agronomic traits for improving the germination and growth of crop plants under drought stress. In this assay, PEG was used to induce osmotic stress on germinating transgenic lines of Arabidopsis thaliana seeds in order to screen for osmotically resistant seed lines.
T2 seeds were plated on BASTA selection plates containing 3% PEG and grown under standard light and temperature conditions. Seeds were plated on each plate containing 3% PEG, ½×MS salts, 1% phytagel, and 10 μg/ml glufosinate. Plates were placed at 4° C. for 3 days to stratify seeds. On day 11, plants were measured for primary root length. After 3 more days of growth, i.e., at day 14, plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was taken on day 14.
Seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success or failure based on whether the plants reached 3 rosette leaves and size of leaves are greater than 1 mm. The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants illustrated in Table 7.
TABLE 7 Growth Seedling Root length Root length stage at weight PEP at day 11 at day 14 day 14 at day 14 SEQ Construct Delta Delta Delta P- Delta ID ID mean P-value mean P-value mean value mean P-value 128 17524 0.468 0.016 0.441 0.026 4.000 / 0.845 0.004 135 18210 0.146 0.027 0.159 0.032 −0.184 0.402 −0.001 0.994 146 18315 0.275 0.025 0.207 0.005 1.235 0.468 0.744 0.058 152 19536 0.252 0.016 0.257 0.033 0.565 0.451 0.256 0.154 155 19614 0.271 0.028 0.138 0.237 4.000 / 0.325 0.043 171 19940 0.262 0.020 0.288 0.010 2.958 0.105 0.422 0.021 179 70938 0.303 0.099 0.256 0.258 4.000 / 0.593 0.038 168 70954 0.198 0.118 0.107 0.460 4.000 / 0.410 0.008 170 70960 0.332 0.019 0.350 0.064 4.000 / 0.399 0.009 174 70978 0.180 0.172 0.092 0.202 4.000 / 0.643 0.003 161 71306 0.284 0.104 0.150 0.277 4.000 / 0.646 0.041 192 72071 0.276 0.006 0.357 0.026 2.587 0.209 −0.033 0.831 199 73493 0.129 0.171 0.142 0.014 1.802 0.291 0.221 0.002 139 75011 0.367 0.001 0.199 0.030 4.000 / 0.691 0.060 147 75080 0.162 0.001 0.107 0.014 1.396 0.068 0.347 0.013 206 75329 0.039 0.690 0.192 0.002 2.406 0.270 0.075 0.674 209 75430 0.105 0.134 0.294 0.038 1.284 0.485 −0.160 0.075 153 76577 0.254 0.224 0.258 0.044 4.000 / 0.381 0.079 195 77605 0.340 0.150 0.445 0.095 2.635 0.061 0.685 0.036 120 78706 0.314 0.061 0.299 0.045 2.973 0.101 0.319 0.000
Transgenic plants comprising recombinant DNA expressing protein as set forth in SEQ ID NO: 118, 122, 126, 142, 173, 196, or 208 showed enhanced PEG osmotic stress tolerance by the second criterial as illustrated in Example 1L and 1M.

F. Cold Shock Tolerance Screen
This example set forth a screen to identify Arabidopsis plants transformed with the genes of interest that are more tolerant to cold stress subjected during day 8 to day 28 after seed planting. During these crucial early stages, seedling growth and leaf area increase were measured to assess tolerance when Arabidopsis seedlings were exposed to low temperatures. Using this screen, genetic alterations can be found that enable plants to germinate and grow better than wild type plants under sudden exposure to low temperatures.
Eleven seedlings from T2 seeds of each transgenic line plus one control line were plated together on a plate containing ½×Gamborg Salts with 0.8 Phytagel™, 1% Phytagel, and 0.3% Sucrose. Plates were then oriented horizontally and stratified for three days at 4° C. At day three, plates were removed from stratification and exposed to standard conditions (16 hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At day eight, plates were removed from standard conditions and exposed to cold shock conditions (24 hr photoperiod, 8° C. at both day and night) until the final day of the assay, i.e., day 28. Rosette areas were measured at day 8 and day 28, which were analyzed as quantitative responses according to example 1M. A list of recombinant nucleotides that improve cold shock stress tolerance in plants is illustrated in Table 8.
TABLE 8 Rosette area Rosette area at day 28 Rosette area PEP at day 8 Risk difference SEQ Construct Delta score Delta ID ID Orientation mean P-value mean P-value mean P-value 122 17520 SENSE 0.764 0.211 1.198 0.021 1.296 0.022 134 17913 SENSE 0.585 0.033 0.403 0.021 0.437 0.000 156 18204 SENSE 0.451 0.099 0.443 0.060 0.452 0.040 140 18507 SENSE 1.039 0.006 1.638 0.003 1.758 0.005 172 70921 SENSE 0.085 0.732 0.157 0.110 0.306 0.033 200 71228 ANTI-SENSE −0.254 0.340 0.677 0.002 1.033 0.002 195 77605 SENSE 0.019 0.949 1.008 0.027 1.017 0.027
Transgenic plants comprising recombinant DNA expressing protein as set forth in SEQ ID NO: 128, 130, or 137 showed enhanced cold stress tolerance by the second criterial as illustrated in Example 1L and 1M.

G. Cold Germination Tolerance Screen
This example sets forth a screen to identify Arabidopsis plants transformed with the genes of interests are resistant to cold stress based on their rate of development, root growth and chlorophyll accumulation under low temperature conditions.
T2 seeds were plated and all seedlings used in the embodiments were grown at 8° C. Seeds were first surface disinfested using chlorine gas and then seeded on assay plates containing an aqueous solution of ½×Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% Phytagel™ (Sigma-Aldrich, P-8169), and 10 ug/ml glufosinate with the final pH adjusted to 5.8 using KOH. Test plates were held vertically for 28 days at a constant temperature of 8° C., a photoperiod of 16 hr, and average light intensity of approximately 100 umol/m 2 /s. At 28 days post plating, root length was measured, growth stage was observed, the visual color was assessed, and a whole plate photograph was taken.
The root length at day 28 was analyzed as a quantitative response according to example 1M. The growth stage at day 7 was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve cold stress tolerance in transgenic plants illustrated in Table 9.
TABLE 9 Root length Growth stage at day 28 at day 28 PEP Construct Nomination Delta Delta SEQ ID ID ID Orientation mean P-value mean P-value 154 11113 CGPG353 ANTI-SENSE 0.180 0.281 4.000 0.000 116 12155 CGPG1135 ANTI-SENSE −0.110 0.202 4.000 0.000 125 17468 CGPG2602 SENSE 0.251 0.075 4.000 0.000 144 18411 CGPG2996 SENSE −0.016 0.839 4.000 0.000 133 18453 CGPG2759 SENSE 0.177 0.247 4.000 0.000 142 18540 CGPG2967 SENSE −0.096 0.749 4.000 0.000 143 19653 CGPG2983 SENSE −0.024 0.911 4.000 0.000 169 19742 CGPG3977 SENSE 0.135 0.292 4.000 0.000 163 70453 CGPG3799 SENSE 0.039 0.660 4.000 0.000 172 70921 CGPG4068 SENSE 0.125 0.029 4.000 0.000 179 70938 CGPG4193 SENSE 0.008 0.955 4.000 0.000 177 70993 CGPG4167 SENSE 0.183 0.027 4.000 0.000 164 71311 CGPG3822 SENSE 0.531 0.008 4.000 0.000 191 72046 CGPG5284 SENSE 0.055 0.721 4.000 0.000 123 72672 CGPG2591 SENSE 0.443 0.101 4.000 0.000 130 72977 CGPG2710 SENSE −0.315 0.347 4.000 0.000 157 73614 CGPG3751 SENSE 0.939 0.009 4.000 0.000 201 74889 CGPG7334 SENSE −0.144 0.648 4.000 0.000 203 74891 CGPG7350 SENSE −0.042 0.860 4.000 0.000 210 75467 CGPG7615 SENSE 0.167 0.308 4.000 0.000 227 77371 CGPG8175 SENSE 0.092 0.530 4.000 0.000
Transgenic plants comprising recombinant DNA expressing protein as set forth in SEQ ID NO: 128, 130, or 137 showed enhanced cold stress tolerance by the second criterial as illustrated in Example 1L and 1M.

H. Shade Tolerance Screen
Plants undergo a characteristic morphological response in shade that includes the elongation of the petiole, a change in the leaf angle, and a reduction in chlorophyll content. While these changes can confer a competitive advantage to individuals, in a monoculture the shade avoidance response is thought to reduce the overall biomass of the population. Thus, genetic alterations that prevent the shade avoidance response can be associated with higher yields. Genes that favor growth under low light conditions can also promote yield, as inadequate light levels frequently limit yield. This protocol describes a screen to look for Arabidopsis plants that show an attenuated shade avoidance response and/or grow better than control plants under low light intensity. Of particular interest, we were looking for plants that didn't extend their petiole length, had an increase in seedling weight relative to the reference and had leaves that were more close to parallel with the plate surface.
T2 seeds were plated on glufosinate selection plates with ½ MS medium. Seeds were sown on ½×MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants were grown on vertical plates at a temperature of 22° C. at day, 20° C. at night and under low light (approximately 30 uE/m 2 /s, far/red ratio (655/665/725/735) ˜0.35 using PLAQ lights with GAM color filter #680). Twenty-three days after seedlings were sown, measurements were recorded including seedling status, number of rosette leaves, status of flower bud, petiole leaf angle, petiole length, and pooled fresh weights. A digital image of the whole plate was taken on the measurement day. Seedling weight and petiole length were analyzed as quantitative responses according to example 1M. The number of rosette leaves, flowering bud formation and leaf angel were analyzed as qualitative responses according to example 1L.
A list of recombinant DNA constructs that improve shade tolerance in plants illustrated in Table 10.








TABLE 10





Seedling weight
Petiole length


at day 23
at day 23










PEP
Construct
Nomination

Delta

Delta


SEQ ID
ID
ID
Orientation
mean
P-value
mean
P-value



160
70458
CGPG3757
SENSE
−0.379
0.068
−0.295
0.091

185
70769
CGPG4622
SENSE
−0.447
0.155
−0.350
0.074

186
71643
CGPG4700
SENSE
0.010
0.950
−0.202
0.047

127
72018
CGPG2651
SENSE
−0.715
0.047
−0.628
0.065

190
72034
CGPG5283
SENSE
−0.709
0.065
−1.001
0.042

129
72078
CGPG2708
SENSE
−1.404
0.006
−1.055
0.045

194
72108
CGPG5322
SENSE
−0.327
0.059
−0.278
0.083

197
73032
CGPG5806
SENSE
−1.540
0.013
−1.286
0.010

181
73216
CGPG4528
SENSE
−0.723
0.016
−0.476
0.041

183
74227
CGPG4560
SENSE
−0.961
0.004
−0.725
0.063

205
75340
CGPG7493
SENSE
−1.326
0.061
−1.053
0.075

209
75430
CGPG7604
SENSE
−0.643
0.086
−0.372
0.092

208
75477
CGPG7600
SENSE
−0.813
0.040
−0.986
0.034

220
75581
CGPG7734
SENSE
−0.679
0.018
−0.616
0.070

222
75658
CGPG7836
SENSE
−0.787
0.003
−0.544
0.034

223
75738
CGPG7867
SENSE
−0.269
0.511
−0.436
0.038

224
77356
CGPG8105
SENSE
−1.669
0.003
−2.412
0.055

215
77822
CGPG7691
SENSE
−0.738
0.112
−0.860
0.027

180
78653
CGPG4210
SENSE
−0.578
0.021
−0.702
0.068

132
78979
CGPG2736
SENSE
−1.116
0.023
−0.736
0.069










































For “seeding weight”, if p<0.05 and delta or risk score mean>0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and delta or risk score mean>0, the transgenic plants showed a trend of trait enhancement as compared to the reference with p<0.2.
For “petiole length”, if p<0.05 and delta<0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and delta<0, the transgenic plants showed a trend of trait enhancement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing protein as set forth in SEQ ID NO: 128, 184, or 195 showed enhanced tolerance to shade or low light condition by the second criterial as illustrated in Example 1L and 1M.
I. Early Plant Growth and Development Screen
This example sets forth a plate based phenotypic analysis platform for the rapid detection of phenotypes that are evident during the first two weeks of growth. In this screen, we were looking for genes that confer advantages in the processes of germination, seedling vigor, root growth and root morphology under non-stressed growth conditions to plants. The transgenic plants with advantages in seedling growth and development were determined by the seedling weight and root length at day 14 after seed planting.
T2 seeds were plated on glufosinate selection plates and grown under standard conditions (˜100 uE/m 2 /s, 16 h photoperiod, 22° C. at day, 20° C. at night). Seeds were stratified for 3 days at 4° C. Seedlings were grown vertically (at a temperature of 22° C. at day 20° C. at night). Observations were taken on day 10 and day 14. Both seedling weight and root length at day 14 were analyzed as quantitative responses according to example 1M.
A list recombinant DNA constructs that improve early plant growth and development illustrated in Table 11.









TABLE 11





Root length
Root length
Seedling weight

PEP

at day 10
at day 14
at day 14












SEQ
Construct
Nomination

Delta

Delta

Delta


ID
ID
ID
Orientation
mean
P-value
mean
P-value
mean
P-value














151
18434
CGPG3454
SENSE
0.188
0.069
0.031
0.585
0.040
0.714

133
18453
CGPG2759
SENSE
0.199
0.149
0.222
0.070
0.061
0.605

178
19762
CGPG4176
SENSE
0.226
0.009
0.133
0.031
0.045
0.835

173
70953
CGPG4105
SENSE
0.347
0.032
0.309
0.052
0.275
0.273

126
75090
CGPG2642
SENSE
0.316
0.020
0.146
0.095
0.459
0.073

211
75457
CGPG7630
SENSE
/
/
/
/
0.373
0.019

213
75470
CGPG7639
SENSE
0.186
0.069
0.128
0.058
0.318
0.039

216
75505
CGPG7696
SENSE
0.135
0.018
0.051
0.567
0.079
0.564

165
76533
CGPG3890
SENSE
0.225
0.075
0.135
0.112
−0.119
0.531


































Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 122, 142, 179, 191 or 195 showed improved early plant growth and development by the second criterial as illustrated in Example 1L and 1M.
J. Late Plant Growth and Development Screen
This example sets forth a soil based phenotypic platform to identify genes that confer advantages in the processes of leaf development, flowering production and seed maturity to plants.
Arabidopsis plants were grown on a commercial potting mixture (Metro Mix 360, Scotts Co., Marysville, Ohio) consisting of 30-40% medium grade horticultural vermiculite, 35-55% sphagnum peat moss, 10-20% processed bark ash, 1-15% pine bark and a starter nutrient charge. Soil was supplemented with Osmocote time-release fertilizer at a rate of 30 mg/ft 3 . T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C. and then sown at a density of ˜5 per 2½″ pot. Thirty-two pots were ordered in a 4 by 8 grid in standard greenhouse flat. Plants were grown in environmentally controlled rooms under a 16 h day length with an average light intensity of ˜200 μmoles/m 2 /s. Day and night temperature set points were 22° C. and 20° C., respectively. Humidity was maintained at 65%. Plants were watered by sub-irrigation every two days on average until mid-flowering, at which point the plants were watered daily until flowering was complete.
Application of the herbicide glufosinate was performed to select T2 individuals containing the target transgene. A single application of glufosinate was applied when the first true leaves were visible. Each pot was thinned to leave a single glufosinate-resistant seedling ˜3 days after the selection was applied.
The rosette radius was measured at day 25. The silique length was measured at day 40. The plant parts were harvested at day 49 for dry weight measurements if flowering production was stopped. Otherwise, the dry weights of rosette and silique were carried out at day 53. The seeds were harvested at day 58. All measurements were analyzed as quantitative responses according to example 1M.
A list of recombinant DNA constructs that improve late plant growth and development illustrated in Table 12.











TABLE 12





Rosette

Seed
Silique




dry
Rosette
net dry
dry
Silique



weight at
radius
weight
weight
length

PEP

day 53
at day 25
at day 62
at day 53
at day 40














SEQ
Construct
Delta
P-
Delta
P-
Delta
P-
Delta
P-
Delta
P-

ID
ID
mean
value
mean
value
mean
value
mean
value
mean
value
















121
72666
0.194
0.032
/
/
0.111
0.267
0.073
0.524
−0.104
0.078

150
18433
−0.363
0.222
0.169
0.006
0.357
0.017
/
/
0.099
0.011

166
70955
−0.137
0.052
0.298
0.003
0.951
0.022
0.163
0.096
−0.001
0.930

161
71306
−0.058
0.556
0.043
0.352
1.522
0.009
0.120
0.011
0.031
0.766

211
75457
−0.147
0.153
0.294
0.055
1.099
0.014
0.496
0.060
−0.217
0.052

212
75469
0.305
0.075
0.267
0.020
1.447
0.015
0.322
0.038
−0.105
0.205

218
75542
1.026
0.018
0.152
0.406
−0.166
0.465
/
/
−0.265
0.174

223
75738
−0.654
0.102
0.064
0.363
0.774
0.000
0.293
0.170
−0.095
0.093



































K. Limited Nitrogen Tolerance Screen
Under low nitrogen conditions, Arabidopsis seedlings become chlorotic and have less biomass. This example sets forth the limited nitrogen tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are altered in their ability to accumulate biomass and/or retain chlorophyll under low nitrogen condition.
T2 seeds were plated on glufosinate selection plates containing 0.5× N-Free Hoagland's T 0.1 mM NH 4 NO 3 T 0.1% sucrose T 1% phytagel media and grown under standard light and temperature conditions. At 12 days of growth, plants were scored for seedling status (i.e., viable or non-viable) and root length. After 21 days of growth, plants were scored for BASTA resistance, visual color, seedling weight, number of green leaves, number of rosette leaves, root length and formation of flowering buds. A photograph of each plant was also taken at this time point.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The number green leaves, the number of rosette leaves and the flowerbud formation were analyzed as qualitative responses according to example 1L. The leaf color raw data were collected on each plant as the percentages of five color elements (Green, DarkGreen, LightGreen, RedPurple, YellowChlorotic) using a computer imaging system. A statistical logistic regression model was developed to predict an overall value based on five colors for each plant.
A list of recombinant DNA constructs that improve low nitrogen availability tolerance in plants illustrated in Table 13.
TABLE 13 Leaf color Root length at day 21 Rosette weight PEP at day 21 Risk at day 21 SEQ Construct Delta score Delta ID ID Orientation mean P-value mean P-value mean P-value 115 10177 ANTI-SENSE −0.556 0.008 3.633 0.013 0.199 0.056 198 11157 SENSE −0.072 0.607 0.928 0.617 0.139 0.051 118 11873 ANTI-SENSE −0.327 0.037 3.065 0.018 −0.013 0.822 117 12233 SENSE / / 2.183 0.087 0.066 0.243 188 12325 ANTI-SENSE −0.366 0.100 1.462 0.027 −0.037 0.588 131 17445 SENSE / / 5.181 0.061 −0.175 0.208 149 18431 SENSE −0.466 0.023 3.868 0.059 −0.021 0.433 184 70678 SENSE −0.084 0.375 1.052 0.035 0.161 0.037 182 71139 SENSE −0.218 0.169 −3.163 0.020 0.490 0.018 186 71643 SENSE 0.028 0.495 −2.371 0.050 0.267 0.046 193 72116 SENSE −0.322 0.074 −0.666 0.093 0.491 0.023 202 74854 SENSE / / 2.673 0.015 −0.051 0.527 203 74891 SENSE −0.403 0.033 1.721 0.022 0.074 0.252 204 75314 SENSE −0.467 0.219 1.983 0.023 −0.160 0.117 209 75430 SENSE −0.429 0.047 5.351 0.011 −0.097 0.352 207 75453 SENSE 0.063 0.684 −1.743 0.341 0.189 0.031 221 75524 SENSE −0.071 0.343 1.084 0.049 −0.089 0.113 218 75542 SENSE −1.768 0.067 3.388 0.029 −0.617 0.132 217 75589 SENSE −0.079 0.443 1.552 0.008 0.011 0.929 226 77363 SENSE / / 3.460 0.065 −0.212 0.086 180 78653 SENSE 0.277 0.214 2.659 0.132 0.203 0.006 136 78710 SENSE / / 3.948 0.007 0.008 0.935 124 78977 SENSE / / 4.985 0.015 0.137 0.118 132 78979 SENSE −0.689 0.220 4.517 0.013 −0.617 0.002
For rosette weight, if p<0.05 and delta or risk score mean>0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and delta or risk score mean>0, the transgenic plants showed a trend of trait enhancement as compared to the reference with p<0.2. For root length, if p<0.05, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2, the transgenic plants showed a trend of trait enhancement as compared to the reference.

L. Statistic Analysis for Qualitative Responses
A list of responses that were analyzed as qualitative responses illustrated in Table 14.







TABLE 14



response
Screen
categories (success vs. failure)



Wilting response Risk
Soil drought tolerance screen
non-wilted vs. wilted

Score

growth stage at day 14
heat stress tolerance screen
50% of plants reach stage 1.03 vs.



not

growth stage at day 14
salt stress tolerance screen
50% of plants reach stage 1.03 vs.



not

growth stage at day 14
PEG induced osmotic stress tolerance
50% of plants reach stage 1.03 vs.


screen
not

growth stage at day 7
cold germination tolerance screen
50% of plants reach stage 0.5 vs. not

number of rosette leaves
Shade tolerance screen
5 leaves appeared vs. not

at day 23

Flower bud formation at
Shade tolerance screen
flower buds appear vs. not

day 23

leaf angle at day 23
Shade tolerance screen
>60 degree vs. <60 degree

number of green leaves at
limited nitrogen tolerance screen
6 or 7 leaves appeared vs. not

day 21

number of rosette leaves
limited nitrogen tolerance screen
6 or 7 leaves appeared vs. not

at day 21

Flower bud formation at
limited nitrogen tolerance screen
flower buds appear vs. not

day 21




































Plants were grouped into transgenic and reference groups and were scored as success or failure according to Table 14. First, the risk (R) was calculated, which is the proportion of plants that were scored as of failure plants within the group. Then the relative risk (RR) was calculated as the ratio of R (transgenic) to R (reference). Risk score (RS) was calculated as −log 2 RR . Two criteria were used to determine a transgenic with enhanced trait(s). Transgenic plants comprising recombinant DNA disclosed herein showed trait enhancement according to either or both of the two criteria.
For the first criteria, the risk scores from multiple events of the transgene of interest were evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc., Cary, N.C., USA). RS with a value greater than 0 indicates that the transgenic plants perform better than the reference. RS with a value less than 0 indicates that the transgenic plants perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference. If p<0.05 and risk score mean>0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean>0, the transgenic plants showed a trend of trait enhancement as compared to the reference.
For the second criteria, the RS from each event was evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The RS with a value greater than 0 indicates that the transgenic plants from this event performs better than the reference. The RS with a value less than 0 indicates that the transgenic plants from this event perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants from this event and the reference don't show any difference. If p<0.05 and risk score mean>0, the transgenic plants from this event showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean>0, the transgenic plants showed a trend of trait enhancement as compared to the reference. If two or more events of the transgene of interest showed improvement in the same response, the transgene was deemed to show trait enhancement.
M. Statistic Analysis for Quantitative Responses
A list of responses that were analyzed as quantitative responses illustrated in Table 15.
TABLE 15 response screen seed yield Soil drought stress tolerance screen seedling weight at day 14 heat stress tolerance screen root length at day 14 heat stress tolerance screen seedling weight at day 14 salt stress tolerance screen root length at day 14 salt stress tolerance screen root length at day 11 salt stress tolerance screen seedling weight at day 14 PEG induced osmotic stress tolerance screen root length at day 11 PEG induced osmotic stress tolerance screen root length at day 14 PEG induced osmotic stress tolerance screen rosette area at day 8 cold shock tolerance screen rosette area at day 28 cold shock tolerance screen difference in rosette area from day 8 to day 28 cold shock tolerance screen root length at day 28 cold germination tolerance screen seedling weight at day 23 Shade tolerance screen petiole length at day 23 Shade tolerance screen root length at day 14 Early plant growth and development screen Seedling weight at day 14 Early plant growth and development screen Rosette dry weight at day 53 Late plant growth and development screen rosette radius at day 25 Late plant growth and development screen seed dry weight at day 58 Late plant growth and development screen silique dry weight at day 53 Late plant growth and development screen silique length at day 40 Late plant growth and development screen Seedling weight at day 21 Limited nitrogen tolerance screen Root length at day 21 Limited nitrogen tolerance screen
The measurements (M) of each plant were transformed by log 2 calculation. The Delta was calculated as log 2 M(transgenic)−log 2 M(reference). Two criteria were used to determine trait enhancement. A transgene of interest could show trait enhancement according to either or both of the two criteria. The measurements (M) of each plant were transformed by log 2 calculation. The Delta was calculated as log 2 M(transgenic)−log 2 M(reference). If the measured response was Petiole Length for the Low Light assay, Delta was subsequently multiplied by −1, to account for the fact that a shorter petiole length is considered an indication of trait enhancement.

For the first criteria, the Deltas from multiple events of the transgene of interest were evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). Delta with a value greater than 0 indicates that the transgenic plants perform better than the reference. Delta with a value less than 0 indicates that the transgenic plants perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference. If p<0.05 and risk score mean>0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean>0, the transgenic plants showed a trend of trait enhancement as compared to the reference.
For the second criteria, the delta from each event was evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc., Cary, N.C., USA). The Delta with a value greater than 0 indicates that the transgenic plants from this event performs better than the reference. The Delta with a value less than 0 indicates that the transgenic plants from this event perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants from this event and the reference don't show any difference. If p<0.05 and delta mean>0, the transgenic plants from this event showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta mean>0, the transgenic plants showed a trend of trait enhancement as compared to the reference. If two or more events of the transgene of interest showed enhancement in the same response, the transgene was deemed to show trait improvement.
Example 2
Identification of Homologs
A BLAST searchable “All Protein Database” is constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a DNA sequence provided herein was obtained, an “Organism Protein Database” is constructed of known protein sequences of the organism; the Organism Protein Database is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.
The All Protein Database is queried using amino acid sequence of cognate protein for gene DNA used in trait-improving recombinant DNA, i.e., sequences of SEQ ID NO: 115 through SEQ ID NO: 228 using “blastp” with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list is kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.
The Organism Protein Database is queried using amino acid sequences of SEQ ID NO: 115 through SEQ ID NO: 228 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits are kept. A BLAST searchable database is constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using “blastp” with E-value cutoff of 1e-8. The hit with the best E-value is compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Likely orthologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 229 to SEQ ID NO: 4815. These orthologs are reported in Tables 16 as homologs to the proteins cognate to genes used in trait-improving recombinant DNA.

















TABLE 16



115:
1365
1361
2178
2823
2234
2825
1854
245
1518
2928
3573
2922


955
1930
1915
2795
467
3944
3288
4383
4338
3604
2927
345


4267
4265
2723
3383
2779
4284
4571
2432
316
310
3031
2099


725
762
442
2727
4758
3997
4738
2662
313
405
1748
3751


4132
3483
1398
3343
2900
397
1254
2642
1508
4249
2573
4178


4424
593
738
595
597
1798
552
923
2444
2389
2396
2740


607
1462

116:
4081
1259
3766
3192
503
1192
4791
698
1068
3223
785
4102

117:
887
4054
4545
665
4519
3562
3980
3020
4522
1726
3155
657


1358
4086
3966
2065
1005
2184
4022
1570
1032

118:
1513
2965
4541
3048
3971
3972
1774
477
4041
2003
3299
3663


1445
2798
1168
4511
1889
4626
3152

119:
2925
2260
3866
4714
2527
3711
4566
2368
1960
3655
1894
2960


4271
2098
2639
539
1297
4222
4224
2811
1914
516
3455
3807


1505
832
1843
1740
4435
240
1919
4235
1867
3639
3240
1861


2797
244
4414
4612
2951
732
1805
3571
3838
2038
3559
1002

120:
2141
365
2225
1364
876
4283
2146
4286
1974
1127
3389
1465


556
2801
4693
273
2062
360
1124
320
341
338
335
292


289
285
283
3419
3557

121:
642
2784
2160
3289
2668
3254
4128
425
1248
2013
4371
4598


2019
1144
820
1036
4619
3449

122:
3177
4253
4257
2808
2789
2433
1394
1339
1333
1336
1335
4636


2178
4187
811
243
2305
2708
2702
4614
2635
2640
2795
2927


3411
3415
723
2829
2111
2103
2653
2628
2625
948
540
4131


4147
4228
4244
4213
305
303
1255
323
2389
1784

123:
3891
574
1849
1832
4018
3244
3777
3780
291
4618
994
965


1028
961
943
939
1568
1122
1119
1095
1025
1010
1006
1044


1077
1043
992
989
3121
356
354
1372
3106
993
390
3391


4210
4252
1630
1813
3431
3636
3161
3585

124:
2583
2651
3877
2863
4243
1233
2941
3494
2914
3154
4793
454


3675
937
380
3067
4551
4057
1220
4251
4588
3970
4474
2864


4032
2654
1455
3380
4351
2633
4809
2410
421
1007
2802
2387

125:
2178
4315
811
815
2370
247
1178
1518
2331
2795
478
3089


2222
2226
2247
2250
3604
2927
2966
3852
1626
4068
2332
1594


4814
2369
2367
3140
2829
1586
4070
3113
899
921
2233
1065


1613
2747
4527
2417
1667
1670
4249
624
745
2445
2929
2787


923
2148
792
323
3071
2348
3810
2444
2389
2740
3404

126:
3322
1820
4486
4040
695
3253
3887
3307
3943
3835
3992
4014


3362
2650
2706
3574
777
1407
1175
3850
4406
2270
4603
2885


947
3776
827
1939
1058
545
466
4658
1812
2011
3736
2139


3057
1551
1230
2469
1828
3750
1397
2563
1191
3441
1278
3531


2403
420
2084
4164
2053
3599

127:
2583
3877
4683
1233
4251
1220
4588
4474
3970
2864
4032
2654


4351
3380
1455
4809
2633
2410
421
1007
4806
3818

128:
401
2840
3369
2530
2805
4475
324
1347
2549
2063
3326
4676

129:
4020
703
2462
2488
1797
991
4489
4582
1351
931
4647
2200


3700
2532
4796
2240
2407
2104
952
1081
3165
3246

130:
3424
2061
3913
259
3775
1236
4394
4028
543
603
1767
2980


3687
998
359
2842
3462
1684
1180
1418
2905
450
3132
1803


3360
638
3859
4066
1291
3917
1295
272

131:
4813
1365
1361
2178
2823
4187
2234
2825
811
815
1518
2050


2640
2635
4144
2795
1942
4572
675
2927
2597
1322
1234
4231


723
3713
3696
1398
4228
1569
863
1819
1667
1670
4249
3968


552
923
303
305
1255
323
2389
1784

132:
1842
1379
3525
3342
1535
1533
3618
1860
3989
3123
1520
1716


2867
271
2627
3010
3747
1534
1530
682
423
3752
3443
3358


4732
559
4350
505
877
3282
4196
1022
3081
4767
1284

133:
1628
480
3341
4415
1152
3236
2385
1074
2303
3429
3551
1161


778
3578
4174
4731
4744
1592
636
402
4378
3139
3540
1659


557
3371
2026
3709
1938
3523
664
3066
2197
4012
2851
1660


3612
4180
3416
3230
1866
981
542
997
1091
2000
653
4289


2870
826
1066

134:
4574
4745
2712
3439
3136
509
1387
1391
1390
1926
1754
3261


3328
1607
1587
1595
1610
3995
3461
3701
1837
3403
3085
3898


4567
2451
3297
1821
2414
4434
3873
1779
2486
1328
1420
1246


1231
673
2261
3259
4532
2878
2164
1084
3765
474
2742

135:
4253
4257
2807
2808
2789
1799
1333
4636
1365
1361
4187
2234


2825
815
811
1405
4245
3243
1518
2995
1506
2635
2640
2331


2795
3633
2264
4573
723
2912
2111
2103
2662
4228
1667
1670


1798
552
923
1255
323
2389
1784
4637
1809

136:
1250
2536
4347
3202
4419
1790
2293
2961
379
2616
1516
3561


1577
764

137:
4402
3160
3677
451
4395
2546
1402
1338
2398
4540
4622
4060


3919
3008
3598
2989
2115
1808
3327
4501
479
858
3055
3718


4804
2602
4413
386
2399
3001
4632
4493
2560
2161
373
1323


1421
3430
428
3211
1060
3305
3311
2952
3458
336
3946
525


3036
643
822

138:
268
3009

139:
3889
4745
3136
1391
1926
1754
3261
3328
1610
1587
2322
3995


893
3461
3701
2564
3197
2534
4510
3848
445
249
1131
2345


4567
2451
3034
1052
660
2613
4799
713
1309
1501
2067
3873


2486
2366
4715
4400
1195
3653
3259
343
367
2164
4392
1084


686
474
3765

140:
1678
1233
3185
4682
3067
4362
1752
964
4339
656
4053
2654


1257
4535
1298
4418
4547
2425
3493
1035
1770
2802
2387
453


2964

141:
2542
1517
1865
4374
1881
3845
1987
2224
4080
1600
557
1588


1207
962
4365
4169
1318
542
2109
997
1091
2000
653
2458


3459
4648
3746

142:
2649
3341
1196
1800
1600
2446
2397
1207
962
4365
4140
4695


542
1091
997
3300
930
3962
1273

143:
1973
2178
2823
815
811
247
1178
1518
2428
1930
1915
2795


3604
2927
1495
1776
552
923
323
2389
914
4158
3414

144:
1143
3272
3274
3315
1854
245
1518
4528
2795
4383
1299
1277


4338
2927
4267
4265
345
2723
3383
4571
2432
4758
534
1748


3751
4132
3483
2900
1254
397
3343
2642
1667
1670
4249
2573


4178
4363
595
552
923
792
2444
2389
2396
759
2740
463

145:
1293
1263
1214
3885
4490
2579
1932
4577
4334
714
3682
280


1416
786
3595
2619
2461
2540
3260
4199
796
4202
4090
528


912
435
3338
632
1900
537
2598
288
2447
4047
2716
1367


501
2151
2720
1564
3507
4299

146:
4508
2457
2498
4463
1198
1202
1522
4137
168
1686
3401
2996


2776
3224
2656
1927
744
795
4726
3222
3183
1434
4447
4500


2015
2854
2839
2836
2852
3789
4157
4737
741
4270
1024
2180


710
4342
4779
3584
3976
3529
1898
1270
2947
3075
3276
4016


2473
2163
262
4564
4134
2330

147:
2494
1573
1553
1591
1589
4051
2323
3221
1699
1702
2525
4319


4139
4141
3017
3016
1680
2994
251
1969
4478
2416
4634
3825


2478
2419
3377
867
1697
1718
1715
1677
1675
2460
2372
1840


3771
4733
1694
1690
1712
4201
3076
2637
2634
794
784
791


813
2604
2360
2529
3124
1113
1989
2463
2479
4304
3465
2978


3799
4550
974
2301
520
1643
1671
1646
3932
4538
644
1846


1901
4613
2971
4808
4298
4359
1321
2289
1316
2953
3356
1783


3252
3472
3107
1751
910
1743
4711
2352
3914
2485
4127
1730


2219
242
586
3080
3102
2813
3602
2792
2483
2559
2553
302


282
3109
1641
536
4264
3473
2576

148:
4792
1080
1818
2039
1864
557
997
1091
2000
653
3444

149:
401
2840
324
2005
1347
2549
1519
2063
4421
3326
1256

150:
3320
634
1294
378
2057
592
617
614
2862
3817
267
1357


1355
1354
1314
4013
2134
408
1994
3091
3629
3111
3593
2313


4497
1484
2048
381
3451
1289
2516
1514
2569
2195
1525
1531


1509
1523
2070
2948
2519
4544
4296
3996
4606
2448
2688
321


3694
589
3670
1481
3902
363
2110
4006
4007
4008
3509
1666


3070
3028
4759
1982
279
1562
3770
1540
3874
524
2296
2241


4368
2210
4259
2129
1090
3830
2832
1123
3518
909
3745
620


4593
3520
4694
635
618
3695

151:
4477
640
213
3024
4491
3762
1929
4645
2609
4215
4484
355


3499
3114
1088
1076
3433
1688
3238
3505
4724
2381
1304
297


4273
506
4689
1448

152:
3320
3756
3758
3699
2028
375
2988
4729
1986
267
1357
1354


1355
1314
3178
4592
4591
3091
4485
3582
3111
3591
4346
4673


2731
580
598
2672
1377
2967
3398
4794
3902
1940
3797
3558


4423
1982
3270
3309
3645
4593
2572
511
4694
4438
2079
747


3040
3983
568
1411
2206
3644

153:
1460
3242
4430
1829
4141
1680
3764
4293
3941
1546
3825
3834


3175
1697
1677
1675
4468
4266
1712
1694
1690
2871
4004
4654


1370
4669
1984
1193
1172
1194
3076
2223
1073
4333
797
813


2360
3799
4550
2728
3975
2301
1671
1643
3669
1578
1901
1096


2276
897
436
3755
1713
3283
3018
4135
3779
2803
266
500


910
2252
1743
753
3163
3162
1730
2219
3256
4183
3367
2689


2765
3623
3437
1641
3610
3144
1887
4264
3805
2853
872
1463

154:
349
1453
4088
1176
2379
2142
1331
946
3969
4707
4552
1841


4153
2942
3482
263
3605
4515
2643
2298
2299
2513
1219
1319


951
326
1549
4025
1026
1027
2799
3271
715
1793
1637
1515


2246
4279
562

155:
2709
4268
1083
3548
4112
1810

156:
3424
3399
2865
4036
2073
543
4505
359
2842
3462
2905
459


2719
4616
3541
3985
1629
2626
783
638
3859
707
4035
1184

157:
988
236
255
301
346
3905
963
1004
4687
296
1009
904


901
879
3928
3929
1162
3927
3912
1046
2600
959
889
885


4401
384
3292
1759
3454
4328
295
3382
2452
766
1863
2760


3524
3503
3485
3506
3836
3714
4611
332
3556
3484
3477
3481


3339
3062
3456
4710
1086
583
2701
2647
2167
2165
2704
2149


2699
3352
3319
3440
3393
3333
3301
3336
3904
3954
4001
3982


3979
3665
3664
3861
3858
3955
3953
3865
3977
3907
3909
3876


3787
3447
3547
3544
3539
3527
2025
625
697
1188
4076
3069


2306
2307
388
2361
234
400
1681
1583
1547
376
4226
2237


2034
3302
3662
2071
2856
1078
1789
1107
3445
3879
3950
3951


3903
3882
3930
3931
3862
3864
3959
3981
3978
3875
4725
4716


1883
1378
2119
1888
1873
1871
4646
1869
986
4713
1816

158:
3729
2266
2722
1410
680
2644
932
3974
2135
1703
2232
891


2242
2238
2265
1647
2190
2269
903
1158
3310
1536
541
1615


4256

159:
988
255
301
346
3905
963
1004
4687
1009
901
879
1162


3927
1145
4145
1835
2320
2600
959
889
885
4401
384
2523


4332
4335
3396
4318
1924
2517
3456
4710
1086
3577
2408
3858


3861
3955
3907
3909
3876
3447
3547
3544
3539
3527
2431
388


2418
1691
4473
3445
3879
3951
3950
3864
3862
3959
3981
3978


3875
4716
4725
1883
986
4765

160:
2166
3361
2042
4437
4450
4706
712
4777
4233
2089
1581

161:
988
236
255
301
963
1004
4687
296
1009
904
901
879


3928
3929
3927
4145
1046
2600
959
889
885
4401
384
3357


2201
2281
2949
717
261
4668
491
3267
4382
3456
4710
1086


2408
3319
3333
3440
3393
3301
3352
3336
3954
3904
3447
3547


3544
3539
3527
1744
4287
388
3516
3354
2386
3452
3683
1078


1789
1107
3445
4716
4725
1883
1378
2119
986
1129

162:
1387
2858
749
4717
3820
746
3837
857
3793
3657
2080
2414


1821
4434
3855
294
2105
4059
886
1217
864
2037
4532
2742


985
4772

163:
760
4005
3088
2102
1435
2243
2847
2845
2587
1899
2262

164:
988
4352
236
255
301
346
3905
963
1004
4687
296
1009


904
901
879
3928
3929
1162
3927
1046
845
959
889
885


816
1075
4151
584
606
560
523
535
504
1653
3382
766


1875
3323
3456
4710
1086
583
3319
3333
3393
3301
3954
3352


3336
3440
3904
3861
3858
3955
3865
3977
3907
3909
3876
3787


3447
3544
3547
3539
3527
4272
2306
2307
388
2361
977
1078


1789
1107
3445
3879
3951
3950
3882
3903
3930
3931
3862
3864


3959
3981
3978
3875
4716
4725
1883
1378
2119
986

165:
1294
375
412
413
267
2562
4013
4485
3591
4673
4346
580


598
4488
4575
1502
2198
2652
2849
1069
3058
3902
3753
1940


3269
393
3509
3410
933
4087
1302
3774
1982
342
337
340


319
4368
663
684
3015
2783
3168
4694
357
4456
484
2475


1419
1807

166:
1363
4115
2693
2690
443
375
971
2343
2346
2351
743
4013


2056
2074
3318
690
3565
2547
553
4061
4063
2112
915
3781


2371
2902
2945
2901
1903
2584
4308
3190
4559
4446
648
2499


1921
1528
958
473
444
446
470
468
465
957
4010
841


395
843
960
1396
3122
3724
2449
2876
3509
1967
253
4445


4558
4368
3702
1951
1464
4674
4595
4118
2349
3520
619
3521


2354
511
742
1735
4143
2100

167:
1325
1725
2821
2903
494
1830
3501
4690
2152
4635
3703
2500


3304
438
1496
4381
4667
4579
4389
4397
4312
419
2255
4514


4741
2899
3988
441
945
1395
2714
2749
2024
873
668
1912


3078
4386
4323
3918
2001
3748
3600
3737
1768
2745
3316
1417


1868
2707
4030
3790
649
772

168:
3730
4508
2457
2498
1438
718
3743
2857
146
2330
4163
582


4354
2209
1950
629
3475
2049
3027
4463
2501
1997
3530
3234


2017
1198
1202
1927
4726
795
3298
748
3222
978
3576
4789


1456
2412
1253
3183
1434
4447
2015
2854
3789
4157
4737
790


3754
1719
1216
1627
1111
1024
741
2180
4270
3584
710
4779


4342
3976
3529
1898
3075
1270
2947
3276
262
2217
4016
2163


4564
2473
4134
4137
1522

169:
2963
1567
3731
2383
572
1266
3698
3280
2318
4454
563
1308


4633
1714
1920
1011
2696
1524
4678
2411
3783
1910
271
3010


1045
836
4666
4186
1432
2130
3317
3872
3432
682
2183
3822


4409
812
4301
433
3634
1454

170:
3688
3255
3188
3167
3073
3072
3690
3235
3150
3097
3094
3092


3090
3715
3200
2168
2032
3594
1527
3174
2325
1372
993
390


3106
3400
825
3588
3984
4252
3636
1630
2308
3431
3161
4045


4701
411
1386
1855
2538
1249
1428
399
1210

171:
1998
4106
3065
488
1753
2054
3533
4114
4219
2916
4077
2259


621
1962
929
4728
2083
391
2521
3606
4357
1003
1498
3345


3022
1565

172:
1403
1548
1545
2273
2963
1379
920
4324
2897
3086
3568
530


4633
1535
1533
3967
2128
1206
1537
1952
2753
733
4171
271


3054
3030
2907
3010
3546
1276
1833
4186
1432
1240
674
2518


1148
2208
456
2076
1616
3538
4396
2155
3324
982
1534
1530


4516
1633
344
2072
3137
2997
565
2524
2991
235
1941
2125


4403
4167
3450
2541
1722
4177
2834
3013
1794
2700
2333
4426


3358
3443
613
631
615
4732
3634
709
3127

173:
878
3586
2171
3138
3794
1345
3844
4699
2279
4015
4801
775


2641
3615
2895
4705
1884
1422
987

174:
2497
1118
1460
1459
3463
3412
4569
2981
2681
3221
2377
4225


2525
2515
1829
4191
3390
3387
3763
2994
251
1479
1975
4478


2416
4634
1544
2476
2478
2424
3376
867
3175
1503
2460
489


490
2372
2392
3047
1608
4733
1747
2871
4004
3560
1965
3460


4229
4227
3044
2634
2192
1073
4333
3832
3064
3061
784
791


3039
2604
2360
1890
2529
2550
3124
1113
3491
462
4685
1136


1989
1013
3465
2978
2979
3799
518
806
3370
3116
3975
4785


4783
4722
4385
1836
2216
2218
2193
705
2191
3308
3871
3961


644
3490
3453
4203
4208
3471
2691
3727
2312
472
2926
2508


1317
2280
711
859
2570
4723
1316
4150
3394
1783
2023
2035


2465
2467
2468
1164
1167
3472
3348
3467
3018
3474
3107
3492


4384
4211
3496
3497
1778
4205
3922
1886
4417
2249
496
2254


700
3914
2352
3163
2485
2484
1362
4786
4780
2674
4784
882


3923
3925
241
1559
586
2043
2138
1593
2812
1429
1376
233


4815
2483
2482
3437
2559
2566
1992
2586
2555
2631
2614
302


282
3045
1831
2438
1785
3148
3142
3473
2337
2275

175:
871
3176
4274
773
1452
1271
4482
1977
4390
4526
4521
1272


1604
2423
4026
3037
276
1905
3042
853
823
4058
510
3880

176:
3424
1334
2204
4055
2472
1597
800
4431
1229
4358
4607
3908


3511
3648
2169
1315
906
4523
4465
4471
2286
848
4017
638


3859
3172

177:
1682
3778
3772
1185
3792
3719
1048
1051
1056
1059
1054
3738


4160
1795
3788
3791
4568
4664
3628
3006
2982
1082
1085
1014


2679
3722
3215
3217
3216
999
300
304
4009
1393
728
2582


4192
2725
2755
2754
3103
1829
3019
4812
4325
4326
1972
1125


4479
689
2622
1433
4064
2777
2775
3941
1546
3313
3194
2127


4355
1000
4773
3627
2405
2880
4753
3671
2101
3366
793
3374


2846
4657
3728
3725
2460
4082
2744
2741
2773
2758
2770
2757


2772
3575
973
968
966
996
1106
1104
1103
1110
2893
2909


2937
1983
1981
3991
1683
2253
2344
4782
4760
4756
4754
3560


1965
605
609
3135
3806
2756
3128
1020
1031
1030
1019
4442


4441
2891
2655
3720
3717
2215
2743
905
908
2186
2202
2088


455
1978
1979
2078
1114
4685
1510
4548
2301
1668
835
849


838
529
1870
4432
3915
4216
2605
299
2892
1204
1201
4749


4380
1166
284
995
2939
2910
1227
1221
1205
3784
404
1468


1265
4002
2686
3960
1781
1780
4049
1171
1186
3526
3392
3549


3987
4031
4052
4065
3099
1225
3281
1676
1622
1580
2228
3478


1679
2010
1695
1701
1674
3257
4416
4649
2739
2736
4677
4665


694
1976
1959
1777
1343
1954
1937
1751
4084
3952
4294
4810


4037
4811
4250
950
2879
869
1693
3936
3935
3934
1288
590


4100
2748
2833
1425
2310
4097
3708
1934
1344
1342
1385
4127


3920
4680
4681
550
722
1943
1945
3651
1787
1197
3901
2665


230
387
2663
2661
2657
3129
2091
3100
1658
1631
1582
1623


1624
1585
1640
1598
1642
1599
1601
1645
1603
2285
1648
1605


1652
3173
4095
913
928
927
924
3115
2581
495
2094
2917


2896
1760
4594
1928
1925
1956
1957
1955
3739
4264
274
3899

178:
2509
4461
1415
3486
3277
3545
3002
2292
512
2822
3619
4138


1079
3896
4803
3635
2898
954
1212
4487
1310
1154
2544
1731


2329
2771
4807
3883
4679
351
3510
4379
4533
3890
1834
2552


513
573
1738
1109
831
2620
4398
881
4204
2561
1826
4050


2012
1443
3079
1003
2113

179:
4608
4453
2712
3439
3136
3295
4212
1406
1409
1371
1404
1754


3261
1587
1610
1612
1607
1595
2322
3995
3219
4405
3096
1450


3461
3701
4091
3824
1213
1897
2615
1174
4567
2451
1478
3033


3808
1427
2887
2884
2889
2924
3813
2095
1307
2067
2154
3259


4532
2878
2164
4392
1084
2677
474
3765
2493
2539
2973

180:
2494
2981
2373
2376
3221
1699
1702
4460
2525
4430
1829
251


2994
2416
3941
1546
3825
2478
2419
2424
3101
3376
2843
2844


3175
1697
1718
1715
1677
1675
1503
2460
4468
2441
3771
1840


4322
4733
1690
1712
1694
4184
3603
4004
4654
1370
4669
4661


3021
1193
1172
1194
1041
1037
1040
1039
4172
4280
1102
4201


3044
3076
2637
2223
797
813
784
2604
2360
2529
3124
4685


1139
2886
1989
1909
3799
4550
3975
4797
704
1671
1643
1646


1661
2188
1247
644
3490
3453
3471
4208
4462
3550
4298
676


315
1944
4723
1316
4277
1783
1882
1190
3472
4524
3467
3018


3107
3492
4211
3496
3497
460
2176
1360
458
701
440
4223


854
3947
3809
4766
4043
1758
910
1743
2957
3209
3208
604


3231
2159
753
3163
2485
3162
2733
884
1730
3256
4183
2765


3602
1948
1466
2559
1121
1773
2553
1634
2438
1742
1737
1641


3610
1887
3473
1827
1970
1359
655
293

181:
2781
461
3916
2746
803
362
3956
2309
4719

182:
4106
2132
1669
193
3196
1512
1497
1050
2766
1498
1099
4218


671
4108
2297

183:
2503
1500
2116
2671
1467
2214
277
1700
3052
1550
824
1880

184:
2883
3007
3827
4079
2045
4104
2137
328
1408
2327
1157
1071


3744
3504
4029
4027
4457
2721
2406
729
1126
1874
330
862


290
2955
4113
2020
1116
1839

185:
3385
2090
4000
1542
2687
2693
2690
2028
443
375
378
412


413
352
1337
2562
3566
610
1098
4343
2454
3611
3608
949


4375
4517
3658
2678
1947
482
4446
2796
2311
2362
2300
3797


4542
957
444
446
958
473
470
468
465
4010
3159
393


2357
2402
1895
389
2145
4410
4407
4443
4425
3640
2830
2008


4445
342
337
340
3512
967
1704
2875
1380
3438
2751
856


4448
4599
2837
2783
3520
4372
4360
4190
2673
2658
511
1584


3108
2762
1745
3112

186:
2132
1532
3533
4219
4114
2916
4077
2046
3878
2510
1146
1072


1003
1498
1099

187:
975
972
3532
3513
4042
1475
1239
532
4704
1261
2522
2287


1656
3831
1286
327
447
911
318
317
3050
3590
2036
3332


687
801
1042
679
616
3378
2006
487
256

188:
3320
3758
3756
4000
2693
2687
2690
4718
375
378
2245
2248


1097
608
2562
610
1098
3178
2134
339
894
1490
2454
4034


3607
4439
4198
4083
4518
2826
1949
699
4446
3753
3159
393


2357
2402
1895
3410
2145
4410
4407
4443
4425
2150
3893
4445


1285
4674
612
1101
4599
2837
2783
4190
2673
2658
278
3105

189:
2494
3158
3625
1460
4569
2981
4051
2373
1699
1702
2395
2525


4430
1829
3035
3023
1680
3764
2356
4483
2416
2415
4634
3941


3825
2478
1392
2419
2421
2436
2424
2394
2843
2844
3175
1718


1697
1715
1677
1675
4643
4570
1503
4468
2378
2441
1840
3771


1712
1690
1694
1766
2871
4184
3603
4654
1370
4004
4669
3560


2064
4660
3003
1193
1172
1194
2645
4072
3220
3076
2223
797


813
2604
1163
2866
3491
252
250
1139
2886
1909
306
1486


2463
2479
3641
3799
4550
2599
3975
2301
4797
704
1671
1643


1646
3669
2196
1247
644
1373
3490
3453
4203
4208
3471
2162


2683
4162
4778
4723
4277
2953
1783
3225
2059
1170
3472
3264


3263
1507
3467
3474
3492
4384
3496
4211
3497
3237
910
1743


3957
3939
3914
2352
2350
2957
3208
753
1349
1382
2578
1730


4183
1555
1556
3367
2765
1593
1376
1651
3602
1948
3437
2559


1121
2555
2553
302
282
1636
3239
3117
2438
2440
1734
1765


1641
3610
1673
4264
3473

190:
2494
3625
4236
4237
3412
2179
4569
2981
4051
3221
1699
1702


2395
3241
4200
2525
4430
4141
4139
3035
1680
2356
4483
2416


2415
4634
1392
2419
2421
2436
2424
2394
3175
1697
1718
1715


1677
1675
4643
4570
1503
2460
2378
2392
2390
2443
2441
1690


1694
1712
4736
2871
3603
4654
1370
2064
4660
1965
1984
3003


1193
1172
1194
2645
4072
3220
3076
2220
813
767
769
3381


2604
2360
2347
3491
252
2890
1579
3641
4304
3799
2732
4625


4275
2763
546
1280
385
1904
3334
4550
4688
4133
3170
2301


4797
704
1643
1671
1646
3669
2894
2496
1985
3958
3961
644


3490
3453
4203
4208
3471
1901
1237
2575
2312
4708
3613
3949


2636
2694
1292
2288
2289
2683
3761
4723
4150
4277
3394
1783


2059
3252
3472
4524
3467
3018
3474
3107
1859
3537
976
2375


3649
3359
799
4502
1203
3564
4601
3492
4384
3496
4211
3497


4805
2278
650
4436
1094
3849
3642
312
1906
2992
457
4019


3408
2009
348
3237
2118
1764
4170
1062
3542
1447
4530
2156


2268
3926
1762
1141
3156
3379
2815
1451
4206
4166
3957
3939


3914
2352
2350
2957
753
2485
1382
2578
1730
2219
4183
720


1555
1556
3080
3102
2765
2689
1376
1117
1268
3602
4815
1948


233
1466
3437
2559
1121
1773
2553
2555
2551
302
282
1636


3239
3117
2438
2440
1734
1641
2778
3144
1303
1673
1887
4264


1301
1327
3473
3418
1572

191:
3422
1173
2271
4411
3077
861
1933
4330
4769
3126
239
809


3314
4290
3508
2703
3668
3291
3716
561
1538
4369
1857
3189


2545
3273
637
2969
3973
3046
1055
602
3210
3212
1961
2507


2505
3397
2667
1663
4345
3193
492
2669
571
984
1689
1705


818
3678
3965
1775
3667
2365
4404
3278
1245
666

192:
2818
2321
3500
2831
2750
2794
2793
309
4735
307
990
821


2087
406
3785
4011
4046
2450
1353
3632
1655
1824
4393
4696


1383
2404
372
3146
4329
1872
1449
1441
1444
1442
2420
3427


953
956
1878
2593
3184
1440
2970
1296
2108
410

193:
4106
2509
434
426
2259
1563
1561
2066
2041
4320
1187
1497


2511
2510
3878
1458
1003
1498
1099
1512
1669

194:
2471
2520
3166
2251
2426
2558
4762
4085
448
1488
2983
3769


2986
3767
4175
3937
4173
4675
2984
3815
4185
3811
2213
2085

195:
1848
3647
860
3534
499
3721
789
1892
3306
2985
1461
2786


3275
3468
3087
437
3800
2030
2477
2873
377
4507
2648
3692


2930
2341
3321
3335
2231
4373
4376
350
4556
4763
4188
4496


4281
322
4103
4742
4476
3782
1935
2624
1650
623
4412
1341


2820
3464
4136
471
4721
1260
1557
3589
3051
2923
4207
870


4408
1664
383
1053
1332
578
1381
1792
1736
2621
577
3228


3804
2007
4336
3005
1575
4387

196:
4129
1692
3621
2205
4429
3145
2353
4159
3290
1431
1437
3833


3180
2768
1034
3656
2277
522
3213
2324
2060
1891
837
808


517
4089
2968
1877
3402
4697
2437
3041
4310
3265
3802
3816


2764
4306
1211
834
1369
1757
2580
2817
907
3118
2456
1100


1252
2711
3661
2427
260
2121
2774
1375
403

197:
2888
2340
2730
1911
2869
1817
3839
4033
1267
659
3856
264


1262
4529
287
936
1366
1008
2824
3130
3428
493
1057
3673


2315
798
3689
4768

198:
1015
3355
4300
275
4344
2120
2676
2284
2990
641
3143
2052


3842
4119
2014
370
4662
4096
4152
1723
432
414
1436
3631


3251
3963
3798
2422
4671
2999
1893
2738
2800
3853
2158
1149


2585
2718
654
3363
3353
2780
2612
850
3933

199:
3795
4165
314
4506
382
3620
4560
3147
4739
3580
4596
1324


4451
1815
1348
1346
661
1763
2710
651
1446
2175
4247
3466


588
3515
1504
364
581
1822
1769
4295
4615
3442
409
1825


2263
2194
1018
254
3293
1696
1771
2124
4509
1093
2504
4056


4553
3583
3630
1224
2400
439

200:
3900
2528
2316
1804
3488
1717
1953
3425
737
1064
1750
449


3487
776
1179
2272
622
4269
2697
2029
4038
4504
392
1971


427
3773
2018
2022
2031
2016
2021
691
1070
527
2737
3514


3819
4422
2548
3659
1644
4311
844
1226
1389
1838

201:
755
2004
3543
4356
752
3384
2274
2153
1609
464
4455
3587


1964
3004
4494
3994
3614
4307
2114
4078
1228
1242
1554
1492


2382
3705
2429
1772
1999
1791
4291
2235
2434
2606
2962

202:
2883
298
4590
852
1038
3786
3828
2267
2670
2933
2491
1071


3744
2759
874
3043
1017
2998
4029
4027
4457
4761
1306
3186


481
2596
3312
1708
1274
1092
916
3011
290
1487
2236

203:
3346
4602
2257
3863
2623
2827
1399
3528
4503
944
498
3676


4800
817
1159
1049
3407
2430
3646
1511
980
1368
567
3406


1412

204:
1403
2093
2273
1326
1483
2908
3596
3342
4633
1520
1716
750


2492
2336
733
1222
4605
4171
526
3010
1602
1706
2931
4186


1432
3686
1120
731
2140
734
1574
3712
768
4168
2187
3634


2211

205:
1687
1632
4146
739
895
890
917
2761
898
2695
896
888


902
883
3757
4317
4321
1638
4628
1238
1401
2002
1067
2838


866
3141
4641
4161
4513
847
4640
3609
2489
3906
248
4638


3684
1786
4651
1029
452
1801
2082
3579
2380
1665
2734
398


3749
2915
3535
4469
1614
1654
4316
3990
1724
1472
4074
761


2913
3368
1287
3554
3337
4650
2591
311
2182
3181
3285
1996


2314
417
2881
507
3164
2291
1142
2577
374
1847
3038
1439


851
3098
2464
4562
1529
3768
3921
3948
3110
4197
1135
3685


3149
833
4230
3622
4659
1012
865
719
3469
2855
416
2841


2782
1047
3735
1279
1782
2919
231
3349
1275
366
3000
4117


2920
4220
3555
3812
2173
2170
2106
942
3691
4492
3638
1016


1811
1474
2646
3674
3581
4182
1424
4388
692
4121
1134
1876


4075
2767
970
652
4670
1621
3498
2906
1806
1199
2339
4642


3843
3169
4495
4427
667
4092
2726
1823
2806
3536
2474
4024


3924
4743
2147
2976
918
4587
1907
2958
3303
3693
3420
1283


2502
258
4440
4123
1374
1560
770
422
4039
1908
469
1470


1756
353
2199
1720
1729
1721
3723
4073
2514
2664
2512
1160


2384
368
551
2675
2848
3153
3851
782
3182
483
1183
693


2804
554
2081
730
1936
4720
3637
2618
3892
2595
3517
3616


4444
3502
4062
1476
3426
4124
1105
1739
3095
2943
702
1414


2068
979
683
246
2659
2334
810
1571
727
4458
558
3895


726
4584
1733
4278
3742
1896
4537
2388
875
3093
925
941


938
2554
940
1662
4467
4452
2868
4655
1223
3205
677
544


2230
2227
569
548
564
566
3867
892
3869
2828
1639
2543


2058
4071
626
2601
555
1269
2453
736
4480
3597
2409
2326


3119
1150
521
3364
1140
658
4044
4399
4179
2932
2752
1685


508
2172
3056
4740
4698
3857
3049
3134
774
3660
1232
2533


1243
2946
486
1732
1244
3421
662
4459
1200
4684
3706
3344


628
2698
575
3759
502
3552
3131
830
1063
3365
3897
724


1400
4214
1620
1182
829
3894
1087
1218
2860
3681
371
2809


4470
3214
2122
1290
519
3284
4348
3592
969
2207
4142
1902


630
415
3248
4554
3519
771
3624
4302
4261
4239
4238
4260


4254
4263
4242
4241
4240
4258
1526
4148
1543
4069
3840
3329


2987
3938

206:
685
1330
1241
1851
4788
2051
4617
2882
1755
4428
1108
3801


2954
2565
1471
1485
4341
716
868
1879
1596
2921
4246
1494


2850
1746
3672
3601

207:
4106
2132
2509
3014
418
4609
2785
2904
633
779
2950
2338


1532
3448
4629
4077
2259
2066
2041
1497
2302
2511
2682
1980


787
2212
485
1138
4361
334
2936
1021
1003
1498
1099
1491


2107
1852

208:
4313
1862
1518
2097
2428
4144
347
4338
3604
2927
2117
4750


4331
316
310
4534
229
4757
3140
2630
534
313
405
3986


1710
1990
4314
1388
3881
2435
2972
4232
4176
639
923
2143


2348
3810
2769
2389
2470

209:
4234
4337
329
4499
3666
533
3331
672
2200
4122
3710
3697


3854
1499
3446
3413
1493
4653
257
3165
4712
2940

210:
1147
265
476
1576
2033
594
3226
3198
1235
1340
4702
696


1539
4585
4285
4364
4546
1590
2638
1208
4764
2713
1480
934


2490
1477
2660
3262
2055
4370
407
2495
1749
3436
3476
2328


3572
2282

211:
1728
3229
3232
4292
3463
3479
2179
3412
4569
2981
2323
3221


1699
1702
4200
2526
3247
3249
1829
4139
4141
3016
3017
1680


4262
3227
3207
1969
4478
2416
4634
4293
1544
2478
1392
3083


3377
867
3175
1697
1718
1677
1675
1503
2460
2392
2872
2874


3047
4266
3771
1840
4733
1712
1690
1694
2871
3560
4201
3044


1617
1619
2632
3832
1073
4333
794
791
784
2610
2604
2592


2360
4149
2529
2550
2535
2531
3124
3491
462
4685
4221
1136


1989
4610
1128
2463
2479
4304
4305
2978
3465
2979
3799
1329


4550
1625
3975
2301
4746
4751
4748
4288
1643
1671
1646
1313


4385
2244
2442
2239
1258
3932
4538
644
3453
3870
1901
4613


2971
3325
2567
2575
431
4193
3206
3201
3203
2819
2590
4734


2288
2537
2788
361
4790
3171
1966
2571
2568
4723
1316
2594


3704
4150
3394
1783
1151
1155
1153
3252
3250
3472
1713
3283


4524
3233
3018
3474
3107
839
840
842
2413
1350
3268
3287


3680
1923
3423
1988
1606
1352
1711
1320
781
3492
4384
1751


1993
4194
4776
4578
880
4391
4703
1115
3294
3435
4795
3841


3914
2352
2481
3163
2959
2485
2484
1362
4127
3846
4747
3643


4774
900
4752
3925
2219
242
1552
586
3080
3102
3082
3084


3367
2043
2138
1593
2813
2342
1651
233
4815
2483
2482
2559


2566
1121
2588
2589
2553
2551
3245
2614
2629
2607
2608
282


302
2221
3847
3195
1831
2438
1788
1641
536
2304
538
3144


1673
4264
1814
3886
3473
4639
4576
585

212:
3386
1913
4531
3068
1473
1264
3279
4481
1796
1946
2791
3191


1112

213:
4689
1448
151
3553
4631
4645
2609
4215
355
4484
587
429


3114
1088
1076
3238
4724
2381
1304
2335
640

214:
3733
3732
3940
3074
2934
3999
3409
4623
2136
3868
4686
2133


4563
4583
4557
2859
4561
2861
4586
4621
3063
475
1858
2040


237
2283
3199
3998
3569
4597
3026
3567
4600
4543
2258
3884


3911
3888
4630
2181
308
1968
4672
2086
2189
814
424
3626


4111
1761
3258
780
2359
611
2935
2975
3457
4023
2131
4620


3563
807
4101
788
2044
627
765
763
2075
4581
2069
2047


4565
2096
4120
4656
828
805
804
4248
2938
3059
2974
4003


3104
2977
3829
1165

215:
4048
678
4464
802
645
4181
4652
2358
325
3204
670
3125


4644
2157
3405
3266
4536
4058
510
1181
4692

216:
681
2506
819
2680
1430
1209
4217
3373
1541
2715
596
2317


2459
2355
2611
3570
1384
1931
983
3741
1423
1958
4297
4512


2439
4498
2393
2363

217:
3320
3385
4000
2693
2687
2690
4718
378
412
2684
3814
4555


352
1337
2562
1311
610
1098
3179
3178
4592
4591
2319
1089


3489
2454
1611
4276
4034
1033
601
4377
4375
4663
1853
1856


3388
2466
2993
514
3910
1169
3395
4154
1133
4446
4420
3753


3797
1895
389
2145
4410
4407
4443
4425
3157
286
735
2144


3340
754
4445
3774
342
337
340
3417
3012
1300
4099
4549


4309
549
4195
3351
4624
3151
2256
4730
1130
430
4674
612


1101
4599
2837
2783
721
846
4372
4360
511
4340
3796

218:
4691
4466
3495
4116
4727
740
4539
3025
1282
3945
4472
3942


570
2790
1963
576
2816
2705
1657

219:
1973
3315
3347
245
1178
1518
3330
3760
4349
1156
1707
1251


751
600
2331
955
1930
1915
2480
2374
4338
497
1917
1918


1916
270
1189
281
3372
2574
316
310
232
4755
3140
313


405
2944
4580
4255
3286
3654
2603
552
599
923
4520
2444


2396
759
1727
3964

220:
4327
1635
1850
4282
4433
3860
1698
2717
2918
3650
1305
2724


706
4209
4021

221:
1001
3296
1845
3522
579
4353
2956
3740
4589
1132
1566
2557


935
1137
669
2126
3726
2685
3707
919
4189
2092
4093
2391


269

222:
1973
4315
4313
4627
3350
1802
531
1709
2123
3617
647
2480


2374
347
4338
3604
3993
3734
4067
2174
1215
1495
1776
229


4534
4757
4781
1672
3140
2630
2662
313
2290
1995
4303
2617


3679
3480
3470
2364
2666
2455
3060
4700
2735
2348
2444
2389


759
2740
2835
2729
3821

223:
2494
4569
4051
1699
1702
3241
2525
4430
1680
251
2994
1482


1479
2416
2415
4634
1544
2478
1392
2419
2424
3376
3175
1718


1697
1715
1677
1675
4643
4570
1503
2460
4733
1690
1712
1694


2871
3560
4280
4201
3044
3076
2634
2637
791
784
813
767


2604
2360
2529
3124
1113
3491
250
252
462
4685
1989
855


1486
3799
1426
4156
4550
3975
2301
2814
1671
1643
1646
1844


3669
644
3490
3453
4208
3471
1618
1901
758
1356
3053
756


1281
3823
3826
2177
1783
3472
1713
3264
3283
3263
1507
3348


3467
2401
1922
3029
646
4802
4211
3496
3497
4798
515
1177


4366
3803
1457
394
1743
4711
3914
2352
2957
3163
2485
2219


2810
2813
2812
1651
4815
1948
3437
2559
1121
2438
2440
1741


1641
1673
3473
2556

224:
2295
4094
4525
1885
1558
396
708
333
331
2203

225:
4787
1413
1023
358
1521
1061
1312
4449
4130

226:
4709
3375
922
926
2077
3218

227:
369
4098
4367
1649
3133
3120
547
1489
2229
2027
591
4604


757
238

228:
3434
1991
2185
3187
4155
4775
2294
3032
2911
1469
4105
4110


4125
4107
4109
3652
4126
2692
4771
2487
688
2877
4770





















































































































































































































































































































































































































































































































































































Example 3
Consensus Sequence Build
ClustalW program is selected for multiple sequence alignments of an amino acid sequence of SEQ ID NO: 115 and its homologs, through SEQ ID NO: 228 and its homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment. The consensus sequence of SEQ ID NO: 181 and its 9 homologs were derived according to the procedure described above and is displayed in FIG. 4 .
Example 4
Pfam Module Annotation
This example illustrates the identification of domain and domain module by Pfam analysis.
The amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam domain modules and individual protein domain for the proteins of SEQ ID NO: 115 through 228 are shown in Table 17 and Table 18 respectively. The Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains. For instance, the protein with amino acids of SEQ ID NO: 118 is characterized by two Pfam domains, i.e. “F-box” and “Tub”. See also the protein with amino acids of SEQ ID NO: 166 which is characterized by two copies of the Pfam domain “Myb_DNA-binding”. In Table 18 “score” is the gathering score for the Hidden Markov Model of the domain which exceeds the gathering cutoff reported in Table 19.








TABLE 17



PEP




Seq
Construct

ID
ID
Pfam Module
Position



115
CGPG110
AP2
79-144

116
CGPG1135
zf-C2H2
126-149

117
CGPG1180
PLATZ
30-141

118
CGPG1197
F-box::Tub
31-87::98-380

119
CGPG1802
zf-C3HC4::YDG_SRA::zf-C3HC4
129-168::253-411::495-551

120
CGPG2561
SBP
62-140

121
CGPG2580
HLH
62-114

122
CGPG2582
AP2
42-106

123
CGPG2591
zf-B_box::zf-B_box
1-47::58-101

124
CGPG2597
zf-C3HC4
111-152

125
CGPG2602
AP2
133-197

126
CGPG2642
HMG_box::HMG_box::HMG_box
138-206::255-321::379-447

127
CGPG2651
zf-C3HC4
102-143

128
CGPG2662
zf-C2H2
152-175

129
CGPG2708
GATA
223-258

130
CGPG2710
HLH
211-261

131
CGPG2722
AP2
68-132

132
CGPG2736
NAM
52-181

133
CGPG2759
zf-Dof
143-205

134
CGPG2765
WRKY
66-125

135
CGPG2802
AP2
42-106

136
CGPG2823
HMG_box
193-260

137
CGPG2889
zf-CCCH::KH_1::zf-CCCH
21-43::95-157::186-211

138
CGPG2902
SRF-TF
14-64

139
CGPG2913
WRKY
222-281

140
CGPG2945
zf-C3HC4
117-158

141
CGPG2962
zf-Dof
35-97

142
CGPG2967
zf-Dof
20-82

143
CGPG2983
AP2
94-158

144
CGPG2996
AP2
83-148

145
CGPG3307
DUF248
95-605

146
CGPG3311
zf-C2H2
89-111

147
CGPG3350
SRF-TF::K-box
9-59::75-173

148
CGPG3355
zf-Dof
24-86

149
CGPG3447
zf-C2H2
117-140

150
CGPG3453
Myb_DNA-binding::Myb_DNA-binding
13-59::65-110

152
CGPG3455
Myb_DNA-binding::Myb_DNA-
75-121::127-173::179-224



binding::Myb_DNA-binding

153
CGPG3488
SRF-TF
9-59

154
CGPG353
Pex2_Pex12::zf-C3HC4
41-270::327-364

155
CGPG3537
bZIP_2
68-122

156
CGPG355
HLH
309-359

157
CGPG3751
GRAS
154-454

158
CGPG3755
Myb_DNA-binding
107-157

159
CGPG3756
GRAS
119-426

160
CGPG3757
F-box
38-85

161
CGPG3776
GRAS
118-437

162
CGPG3778
WRKY
109-171

163
CGPG3799
AT_hook::DUF296
76-88::160-280

164
CGPG3822
GRAS
248-550

165
CGPG3890
Myb_DNA-binding::Myb_DNA-binding
105-151::157-202

166
CGPG3965
Myb_DNA-binding::Myb_DNA-binding
17-66::72-117

167
CGPG3966
HLH
74-125

168
CGPG3974
zf-C2H2
94-116

169
CGPG3977
NAM
11-135

170
CGPG3984
zf-B_box::zf-B_box
1-47::52-99

171
CGPG3988
Myb_DNA-binding
98-145

172
CGPG4068
NAM
7-132

173
CGPG4105
PHD
198-248

174
CGPG4109
SRF-TF::K-box
9-59::75-175

175
CGPG4125
zf-C3HC4
156-196

176
CGPG4126
HLH
129-178

177
CGPG4167
SRF-TF::K-box
9-59::71-173

178
CGPG4176
Myb_DNA-binding
9-58

179
CGPG4193
WRKY
243-303

180
CGPG4210
SRF-TF::K-box
9-59::78-174

182
CGPG4540
Myb_DNA-binding
94-141

184
CGPG4571
Myb_DNA-binding::Linker_histone
5-57::126-201

185
CGPG4622
Myb_DNA-binding::Myb_DNA-binding
12-59::65-110

186
CGPG4700
Myb_DNA-binding::Myb_DNA-binding
5-56::134-181

187
CGPG475
bZIP_1
89-142

188
CGPG478
Myb_DNA-binding::Myb_DNA-binding
14-61::67-112

189
CGPG497
SRF-TF::K-box
9-59::74-173

190
CGPG5283
SRF-TF::K-box
25-75::88-189

191
CGPG5284
CBFD_NFYB_HMF
102-166

192
CGPG5294
AUX_IAA
11-192

193
CGPG5308
Myb_DNA-binding::Myb_DNA-binding
11-60::117-164

194
CGPG5322
LIM::LIM
11-68::110-167

195
CGPG5427
IBR
208-271

196
CGPG5595
SET
746-874

197
CGPG5806
bZIP_1
169-233

198
CGPG626
Mov34
54-168

199
CGPG6434
ZZ::Myb_DNA-binding::SWIRM
1-46::62-108::349-434

200
CGPG688
bZIP_1
187-245

201
CGPG7334
E2F_TDP::E2F_TDP
12-77::148-224

202
CGPG7339
Myb_DNA-binding
5-57

203
CGPG7350
Prefoldin
24-143

204
CGPG7475
NAM
18-147

205
CGPG7493
HD-ZIP_N::Homeobox::HALZ
1-115::141-195::196-240

207
CGPG7598
Myb_DNA-binding::Myb_DNA-binding
30-79::136-183

208
CGPG7600
AP2
20-84

209
CGPG7604
GATA
212-247

210
CGPG7615
zf-C3HC4
150-187

211
CGPG7630
SRF-TF::K-box
9-59::75-174

212
CGPG7631
bZIP_1
121-178

214
CGPG7644
TCP
38-253

215
CGPG7691
zf-C3HC4
30-76

216
CGPG7696
Ank::Ank::zf-CCCH::zf-CCCH
77-112::114-149::270-295




::305-327

217
CGPG7703
Myb_DNA-binding::Myb_DNA-binding
14-61::67-112

218
CGPG7707
TCP
16-209

219
CGPG7731
AP2
111-176

220
CGPG7734
ZZ
307-360

221
CGPG7753
zf-C2H2::zf-C2H2
62-84::142-164

222
CGPG7836
AP2
23-87

223
CGPG7867
SRF-TF::K-box
9-59::85-175

224
CGPG8105
B3
128-225

225
CGPG8132
zf-LSD1::zf-LSD1
63-87::101-125

227
CGPG8175
HLH
86-135










































































































































TABLE 18



PEP Seq
Construct
Pfam domain





ID No.
ID
name
begin
start
score
E-value













115
CGPG110
AP2
79
144
152.7
9.50E−43

116
CGPG1135
zf-C2H2
126
149
19.8
0.0097

117
CGPG1180
PLATZ
30
141
288.2
1.60E−83

118
CGPG1197
F-box
31
87
36.1
1.20E−07

118
CGPG1197
Tub
98
380
627.7
1.00E−185

119
CGPG1802
zf-C3HC4
129
168
36.9
6.90E−08

119
CGPG1802
YDG_SRA
253
411
259.6
6.20E−75

119
CGPG1802
zf-C3HC4
495
551
28.9
1.70E−05

120
CGPG2561
SBP
62
140
187.5
3.10E−53

121
CGPG2580
HLH
62
114
67
6.30E−17

122
CGPG2582
AP2
42
106
148.8
1.50E−41

123
CGPG2591
zf-B_box
1
47
39.8
9.50E−09

123
CGPG2591
zf-B_box
58
101
44.3
4.20E−10

124
CGPG2597
zf-C3HC4
111
152
36.5
9.20E−08

125
CGPG2602
AP2
133
197
138.5
1.80E−38

126
CGPG2642
HMG_box
138
206
71.4
2.90E−18

126
CGPG2642
HMG_box
255
321
84.2
3.90E−22

126
CGPG2642
HMG_box
379
447
77.6
4.00E−20

127
CGPG2651
zf-C3HC4
102
143
39.9
9.00E−09

128
CGPG2662
zf-C2H2
152
175
20.1
0.0078

129
CGPG2708
GATA
223
258
67.8
3.50E−17

130
CGPG2710
HLH
211
261
32.6
1.40E−06

131
CGPG2722
AP2
68
132
138.3
2.10E−38

132
CGPG2736
NAM
52
181
56.9
6.80E−14

133
CGPG2759
zf-Dof
143
205
132.1
1.60E−36

134
CGPG2765
WRKY
66
125
141.6
2.10E−39

135
CGPG2802
AP2
42
106
135.2
1.80E−37

136
CGPG2823
HMG_box
193
260
32.9
1.20E−06

137
CGPG2889
zf-CCCH
21
43
4.4
0.35

137
CGPG2889
KH_1
95
157
58.8
1.70E−14

137
CGPG2889
zf-CCCH
186
211
46
1.30E−10

138
CGPG2902
SRF-TF
14
64
29.5
1.20E−05

139
CGPG2913
WRKY
222
281
144.4
3.00E−40

140
CGPG2945
zf-C3HC4
117
158
21.3
0.00098

141
CGPG2962
zf-Dof
35
97
134.3
3.40E−37

142
CGPG2967
zf-Dof
20
82
126.1
9.60E−35

143
CGPG2983
AP2
94
158
75.7
1.50E−19

144
CGPG2996
AP2
83
148
137.1
4.90E−38

145
CGPG3307
DUF248
95
605
1274.5
0

146
CGPG3311
zf-C2H2
89
111
23
0.001

147
CGPG3350
SRF-TF
9
59
106.1
1.00E−28

147
CGPG3350
K-box
75
173
108.1
2.60E−29

148
CGPG3355
zf-Dof
24
86
139.1
1.20E−38

149
CGPG3447
zf-C2H2
117
140
20.2
0.0074

150
CGPG3453
Myb_DNA-binding
13
59
60
7.50E−15

150
CGPG3453
Myb_DNA-binding
65
110
48.9
1.70E−11

152
CGPG3455
Myb_DNA-binding
75
121
54.7
3.00E−13

152
CGPG3455
Myb_DNA-binding
127
173
59.3
1.30E−14

152
CGPG3455
Myb_DNA-binding
179
224
44.6
3.40E−10

153
CGPG3488
SRF-TF
9
59
66.2
1.00E−16

154
CGPG353
Pex2_Pex12
41
270
288.3
1.40E−83

154
CGPG353
zf-C3HC4
327
364
29.3
1.30E−05

155
CGPG3537
bZIP_1
68
132
28.3
2.80E−05

155
CGPG3537
bZIP_2
68
122
47.8
3.70E−11

156
CGPG355
HLH
309
359
32.8
1.20E−06

157
CGPG3751
GRAS
154
454
524.7
1.00E−154

158
CGPG3755
Myb_DNA-binding
107
157
43.6
6.50E−10

159
CGPG3756
GRAS
119
426
385.6
7.60E−113

160
CGPG3757
F-box
38
85
43.3
8.20E−10

161
CGPG3776
GRAS
118
437
450.8
1.80E−132

162
CGPG3778
WRKY
109
171
93.5
6.60E−25

163
CGPG3799
AT_hook
76
88
19.1
0.012

163
CGPG3799
DUF296
160
280
218.5
1.50E−62

164
CGPG3822
GRAS
248
550
469.7
3.70E−138

165
CGPG3890
Myb_DNA-binding
105
151
54.9
2.70E−13

165
CGPG3890
Myb_DNA-binding
157
202
46.9
6.80E−11

166
CGPG3965
Myb_DNA-binding
17
66
44.9
2.80E−10

166
CGPG3965
Myb_DNA-binding
72
117
39.8
9.30E−09

167
CGPG3966
HLH
74
125
39.7
9.70E−09

168
CGPG3974
zf-C2H2
94
116
22.4
0.0016

169
CGPG3977
NAM
11
135
260.6
3.10E−75

170
CGPG3984
zf-B_box
1
47
51
4.00E−12

170
CGPG3984
zf-B_box
52
99
58.5
2.20E−14

171
CGPG3988
Myb_DNA-binding
98
145
40.9
4.30E−09

172
CGPG4068
NAM
7
132
301.1
2.10E−87

173
CGPG4105
PHD
198
248
56.1
1.10E−13

174
CGPG4109
SRF-TF
9
59
116.6
7.00E−32

174
CGPG4109
K-box
75
175
147.1
4.50E−41

175
CGPG4125
zf-C3HC4
156
196
27.9
3.50E−05

176
CGPG4126
HLH
129
178
35.9
1.40E−07

177
CGPG4167
SRF-TF
9
59
113.7
5.40E−31

177
CGPG4167
K-box
71
173
74.5
3.40E−19

178
CGPG4176
Myb_DNA-binding
9
58
31.5
2.90E−06

179
CGPG4193
WRKY
243
303
143.4
6.20E−40

180
CGPG4210
SRF-TF
9
59
104.2
3.70E−28

180
CGPG4210
K-box
78
174
44.1
4.60E−10

182
CGPG4540
Myb_DNA-binding
94
141
47.2
5.70E−11

184
CGPG4571
Myb_DNA-binding
5
57
33.4
8.20E−07

184
CGPG4571
Linker_histone
126
201
25.8
2.60E−05

185
CGPG4622
Myb_DNA-binding
12
59
40.5
5.60E−09

185
CGPG4622
Myb_DNA-binding
65
110
41.6
2.70E−09

186
CGPG4700
Myb_DNA-binding
5
56
39.7
1.00E−08

186
CGPG4700
Myb_DNA-binding
134
181
43.9
5.50E−10

187
CGPG475
bZIP_1
89
142
28.7
2.00E−05

187
CGPG475
bZIP_2
89
139
17.9
0.011

188
CGPG478
Myb_DNA-binding
14
61
48.8
1.90E−11

188
CGPG478
Myb_DNA-binding
67
112
48.5
2.30E−11

189
CGPG497
SRF-TF
9
59
121
3.30E−33

189
CGPG497
K-box
74
173
150.5
4.60E−42

190
CGPG5283
SRF-TF
25
75
118.8
1.60E−32

190
CGPG5283
K-box
88
189
160
6.00E−45

191
CGPG5284
Histone
96
169
53.2
8.70E−13

191
CGPG5284
CBFD_NFYB_HMF
102
166
93.4
6.90E−25

192
CGPG5294
AUX_IAA
11
192
391.1
1.60E−114

193
CGPG5308
Myb_DNA-binding
11
60
22.5
0.0016

193
CGPG5308
Myb_DNA-binding
117
164
48.6
2.10E−11

194
CGPG5322
LIM
11
68
53.4
7.70E−13

194
CGPG5322
LIM
110
167
63.9
5.20E−16

195
CGPG5427
IBR
208
271
85
2.30E−22

196
CGPG5595
SET
746
874
159.9
6.70E−45

197
CGPG5806
bZIP_1
169
233
28.2
2.80E−05

197
CGPG5806
bZIP_2
169
223
24.7
0.00033

198
CGPG626
Mov34
54
168
178.6
1.50E−50

199
CGPG6434
ZZ
1
46
85.2
2.00E−22

199
CGPG6434
Myb_DNA-binding
62
108
48.1
3.10E−11

199
CGPG6434
SWIRM
349
434
83.2
7.90E−22

200
CGPG688
bZIP_1
187
245
46
1.30E−10

200
CGPG688
bZIP_2
188
241
44.6
3.30E−10

201
CGPG7334
E2F_TDP
12
77
115.1
2.00E−31

201
CGPG7334
E2F_TDP
148
224
119
1.30E−32

202
CGPG7339
Myb_DNA-binding
5
57
35
2.60E−07

203
CGPG7350
Prefoldin
24
143
126.1
9.60E−35

204
CGPG7475
NAM
18
147
257.9
2.00E−74

205
CGPG7493
HD-ZIP_N
1
115
139.2
1.10E−38

205
CGPG7493
Homeobox
141
195
72
1.90E−18

205
CGPG7493
HALZ
196
240
89.5
1.00E−23

207
CGPG7598
Myb_DNA-binding
30
79
48.6
2.10E−11

207
CGPG7598
Myb_DNA-binding
136
183
49.6
1.10E−11

208
CGPG7600
AP2
20
84
143.2
6.90E−40

209
CGPG7604
GATA
212
247
67.2
5.30E−17

210
CGPG7615
zf-C3HC4
150
187
33.4
7.70E−07

211
CGPG7630
SRF-TF
9
59
111.6
2.20E−30

211
CGPG7630
K-box
75
174
157.8
2.80E−44

212
CGPG7631
bZIP_1
121
178
30.1
7.80E−06

212
CGPG7631
bZIP_2
121
175
26.1
0.00013

214
CGPG7644
TCP
38
253
139.2
1.10E−38

215
CGPG7691
zf-C3HC4
30
76
28.2
2.90E−05

216
CGPG7696
Ank
77
112
13.1
0.4

216
CGPG7696
Ank
114
149
31.7
2.60E−06

216
CGPG7696
zf-CCCH
270
295
27.2
6.00E−05

216
CGPG7696
zf-CCCH
305
327
0.4
1.2

217
CGPG7703
Myb_DNA-binding
14
61
51.5
2.80E−12

217
CGPG7703
Myb_DNA-binding
67
112
34.7
3.10E−07

218
CGPG7707
TCP
16
209
149.1
1.20E−41

219
CGPG7731
AP2
111
176
146.8
5.70E−41

220
CGPG7734
ZZ
307
360
27
6.50E−05

221
CGPG7753
zf-C2H2
62
84
27.3
5.30E−05

221
CGPG7753
zf-C2H2
142
164
23.6
0.00071

222
CGPG7836
AP2
23
87
134.4
3.20E−37

223
CGPG7867
SRF-TF
9
59
95.2
1.90E−25

223
CGPG7867
K-box
85
175
22.5
5.60E−06

224
CGPG8105
B3
128
225
82.2
1.60E−21

225
CGPG8132
zf-LSD1
63
87
50.1
7.30E−12

225
CGPG8132
zf-LSD1
101
125
54.5
3.50E−13

227
CGPG8175
HLH
86
135
32.2
1.80E−06
























































































































































































TABLE 19





gathering


Pfam domain name
Accession #
cutoff
domain description










AT_hook
PF02178.9
3.6
AT hook motif

AUX_IAA
PF02309.7
−83
AUX/IAA family

Ank
PF00023.20
0
Ankyrin repeat

B3
PF02362.12
26.5
B3 DNA binding domain

CBFD_NFYB_HMF
PF00808.13
18.4
Histone-like transcription factor (CBF/NF-Y) and




archaeal histone

DUF248
PF03141.7
−363.8
Putative methyltransferase

DUF296
PF03479.5
−11
Domain of unknown function (DUF296)

E2F_TDP
PF02319.11
17
E2F/DP family winged-helix DNA-binding domain

F-box
PF00646.23
13.9
F-box domain

GATA
PF00320.17
28.5
GATA zinc finger

GRAS
PF03514.5
−78
GRAS family transcription factor

HALZ
PF02183.8
17
Homeobox associated leucine zipper

HD-ZIP_N
PF04618.3
25
HD-ZIP protein N terminus

HLH
PF00010.16
8.3
Helix-loop-helix DNA-binding domain

HMG_box
PF00505.9
4.1
HMG (high mobility group) box

Histone
PF00125.14
17.4
Core histone H2A/H2B/H3/H4

Homeobox
PF00046.19
−4.1
Homeobox domain

IBR
PF01485.11
0
IBR domain

K-box
PF01486.8
0
K-box region

KH_1
PF00013.19
10.5
KH domain

LIM
PF00412.12
0
LIM domain

Linker_histone
PF00538.9
−8
linker histone H1 and H5 family

Mov34
PF01398.11
−4
Mov34/MPN/PAD-1 family

Myb_DNA-binding
PF00249.21
14
Myb-like DNA-binding domain

NAM
PF02365.6
−19
No apical meristem (NAM) protein

PHD
PF00628.19
25.9
PHD-finger

PLATZ
PF04640.4
20
PLATZ transcription factor

Pex2_Pex12
PF04757.5
−50
Pex2/Pex12 amino terminal region

Prefoldin
PF02996.8
9.3
Prefoldin subunit

SBP
PF03110.5
25
SBP domain

SET
PF00856.18
23.5
SET domain

SRF-TF
PF00319.9
11
SRF-type transcription factor (DNA-binding and




dimerisation domain)

SWIRM
PF04433.7
25
SWIRM domain

TCP
PF03634.4
−38
TCP family transcription factor

Tub
PF01167.8
−98
Tub family

WRKY
PF03106.6
25
WRKY DNA-binding domain

YDG_SRA
PF02182.8
25
YDG/SRA domain

ZZ
PF00569.8
14
Zinc finger, ZZ type

bZIP_1
PF00170.11
24.5
bZIP transcription factor

bZIP_2
PF07716.5
15
Basic region leucine zipper

zf-B_box
PF00643.14
15.3
B-box zinc finger

zf-C2H2
PF00096.16
17.7
Zinc finger, C2H2 type

zf-C3HC4
PF00097.15
16
Zinc finger, C3HC4 type (RING finger)

zf-CCCH
PF00642.15
0
Zinc finger C-x8-C-x5-C-x3-H type (and similar)

zf-Dof
PF02701.6
25
Dof domain, zinc finger

zf-LSD1
PF06943.3
25
LSD1 zinc finger





































































Example 5
Plasmid Construction for Transferring Recombinant DNA
This example illustrates the construction of plasmids for transferring recombinant DNA into the nucleus of a plant cell which can be regenerated into a transgenic crop plant of this invention. Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. DNA of interest, i.e. each DNA identified in Table 2 and the DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the base vector.
A. Plant Expression Constructs for Corn Transformation
Elements of an exemplary common expression vector, pMON93039 are illustrated in Table 20. The exemplary base vector which is especially useful for corn transformation is illustrated in FIG. 2 and assembled using technology known in the art.





TABLE 20



pMON93039









Coordinates of




SEQ ID NO:

function
name
annotation
4816




Agrobacterium

B-AGRtu.right border
Agro right border
11364-11720

T-DNA

sequence, essential for

trabsfer

transfer of T-DNA.

Gene of
E-Os.Act1
upstream promoter
19-775

interest

region of the rice actin

expression

1 gene

cassette
E-CaMV.35S.2xA1-B3
duplicated35S A1-B3
788-1120



domain without TATA



box


P-Os.Act1
promoter region of the
1125-1204



rice actin 1 gene


L-Ta.Lhcb1
5′ untranslated leader
1210-1270



of wheat major



chlorophyll a/b binding



protein


I-Os.Act1
first intron and flanking
1287-1766



UTR exon sequences



from the rice actin 1



gene


T-St.Pis4
3′ non-translated region
1838-2780



of the potato proteinase



inhibitor II gene which



functions to direct



polyadenylation of the



mRNA

Plant
P-Os.Act1
Promoter from the rice
2830-3670

selectable

actin 1 gene

marker
L-Os.Act1
first exon of the rice
3671-3750

expression

actin 1 gene

cassette
I-Os.Act1
first intron and flanking
3751-4228



UTR exon sequences



from the rice actin 1



gene


TS-At.ShkG-CTP2
Transit peptide region
4238-4465



of Arabidopsis EPSPS


CR-AGRtu.aroA-
coding region for
4466-5833


CP4.nat
bacterial strain CP4



native arogA gene


T-AGRtu.nos
A 3′ non-translated
5849-6101



region of the nopaline



synthase gene of




Agrobacterium




tumefaciens Ti plasmid



which functions to



direct polyadenylation



of the mRNA.


Agrobacterium

B-AGRtu.left border
Agro left border
6168-6609

T-DNA

sequence, essential for

transfer

transfer of T-DNA.

Maintenance
OR-Ec.oriV-RK2
The vegetative origin
6696-7092

in E. coli

of replication from



plasmid RK2.


CR-Ec.rop
Coding region for
8601-8792



repressor of primer



from the ColE1



plasmid. Expression of



this gene product



interferes with primer



binding at the origin of



replication, keeping



plasmid copy number



low.


OR-Ec.ori-ColE1
The minimal origin of
9220-9808



replication from the E. coli



plasmid ColE1.


P-Ec.aadA-SPC/STR
romoter for Tn7
10339-10380



adenylyltransferase



(AAD(3″))


CR-Ec.aadA-SPC/STR
Coding region for Tn7
10381-11169



adenylyltransferase



(AAD(3″)) conferring



spectinomycin and



streptomycin



resistance.


T-Ec.aadA-SPC/STR
3′ UTR from the Tn7
11170-11227



adenylyltransferase



(AAD(3″)) gene of E. coli .



































































































B. Plant Expression Constructs for Soybean or Canola Transformation
Plasmids for use in transformation of soybean are also prepared. Elements of an exemplary common expression vector plasmid pMON82053 are shown in Table 21 below. This exemplary soybean transformation base vector illustrated in FIG. 2 is assembled using the technology known in the art. DNA of interest, i.e. each DNA identified in Table 2 and the DNA for the identified homologous genes, is cloned and amplified by PCR prior to insertion into the insertion site the base vector at the insertion site between the enhanced 35S CaMV promoter and the termination sequence of cotton E6 gene.





TABLE 21



pMON82053









Coordinates of




SEQ ID NO:

function
name
annotation
4817




Agrobacterium

B-AGRtu.left
Agro left border sequence, essential
6144-6585

T-DNA
border
for transfer of T-DNA.

transfer

Plant
P-At.Act7
Promoter from the arabidopsis actin
6624-7861

selectable

7 gene

marker
L-At.Act7
5′UTR of Arabidopsis Act7 gene

expression
I-At.Act7
Intron from the Arabidopsis actin7

cassette

gene


TS-At.ShkG-
Transit peptide region of Arabidopsis
7864-8091


CTP2
EPSPS


CR-AGRtu.aroA-
Synthetic CP4 coding region with
8092-9459


CP4.nno_At
dicot preferred codon usage.


T-AGRtu.nos
A 3′ non-translated region of the
9466-9718



nopaline synthase gene of



Agrobacterium tumefaciens Ti



plasmid which functions to direct



polyadenylation of the mRNA.

Gene of
P-CaMV.35S-enh
Promoter for 35S RNA from CaMV
1-613

interest

containing a duplication of the −90 to

expression

−350 region.

cassette
T-Gb.E6-3b
3′ untranslated region from the fiber
688-1002



protein E6 gene of sea-island cotton;


Agrobaterium

B-AGRtu.right
Agro right border sequence, essential
1033-1389

T-DNA
border
for transfer of T-DNA.

transfer

Maintenance
OR-Ec.oriV-RK2
The vegetative origin of replication
5661-6057

in E. coli

from plasmid RK2.


CR-Ec.rop
Coding region for repressor of
3961-4152



primer from the ColE1 plasmid.



Expression of this gene product



interferes with primer binding at the



origin of replication, keeping



plasmid copy number low.


OR-Ec.ori-ColE1
The minimal origin of replication
2945-3533



from the E. coli plasmid ColE1.


P-Ec.aadA-
romoter for Tn7 adenylyltransferase
2373-2414


SPC/STR
(AAD(3″))


CR-Ec.aadA-
Coding region for Tn7
1584-2372


SPC/STR
adenylyltransferase (AAD(3″))



conferring spectinomycin and



streptomycin resistance.


T-Ec.aadA-
3′ UTR from the Tn7
1526-1583


SPC/STR
adenylyltransferase (AAD(3″)) gene



of E. coli .


































































C. Plant Expression Constructs for Cotton Transformation
Plasmids for use in transformation of cotton are also prepared. Elements of an exemplary common expression vector plasmid pMON99053 are shown in Table 22 below and FIG. 3 . Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Each recombinant DNA coding for a protein identified in Table 2 is amplified by PCR prior to insertion into the insertion site within the gene of interest expression cassette of one of the base.





TABLE 22



pMON99053









Coordinates




of SEQ ID

function
name
annotation
NO: 4818




Agrobacterium

B-AGRtu.right
Agro right border sequence,
1-357

T-DNA transfer
border
essential for transfer of T-



DNA.

Gene of interest
Exp-CaMV.35S-
Enhanced version of the 35S
388-1091

expression cassette
enh + ph.DnaK
RNA promoter from CaMV



plus the petunia hsp70 5′



untranslated region


T-Ps.RbcS2-E9
The 3′ non-translated region of
1165-1797



the pea RbcS2 gene which



functions to direct



polyadenylation of the mRNA.

Plant selectable
Exp-CaMV.35S
Promoter and 5′ untranslated
1828-2151

marker expression

region of the 35S RNA from

cassette

CaMV


CR-Ec.nptII-Tn5
Neomycin Phosphotransferase
2185-2979



II gene that confers resistance



to neomycin and kanamycin


T-AGRtu.nos
A 3′ non-translated region of
3011-3263



the nopaline synthase gene of



Agrobacterium tumefaciens Ti



plasmid which functions to



direct polyadenylation of the



mRNA.


Agrobacterium

B-AGRtu.left
Agro left border sequence,
3309-3750

T-DNA transfer
border
essential for transfer of T-



DNA.

Maintenance in E. coli
OR-Ec.oriV-
The vegetative origin of
3837-4233


RK2
replication from plasmid RK2.


CR-Ec.rop
Coding region for repressor of
5742-5933



primer from the ColE1



plasmid. Expression of this



gene product interferes with



primer binding at the origin of



replication, keeping plasmid



copy number low.


OR-Ec.ori-
The minimal origin of
6361-6949


ColE1
replication from the E. coli



plasmid ColE1.


P-Ec.aadA-
romoter for Tn7
7480-7521


SPC/STR
adenylyltransferase (AAD(3″))


CR-Ec.aadA-
Coding region for Tn7
7522-8310


SPC/STR
adenylyltransferase (AAD(3″))



conferring spectinomycin and



streptomycin resistance.


T-Ec.aadA-
3′ UTR from the Tn7
8311-8368


SPC/STR
adenylyltransferase (AAD(3″))



gene of E. coli .





































































Example 6
Corn Plant Transformation
This example illustrates the production and identification of transgenic corn cells in seed of transgenic corn plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or enhanced seed compositions as compared to control plants. Transgenic corn cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 2 by Agrobacterium -mediated transformation using the corn transformation constructs as disclosed in Example 5.
Corn transformation is effected using methods disclosed in U.S. Patent Application Publication 2004/0344075 A1 where corn embryos are inoculated and co-cultured with the Agrobacterium tumefaciens strain ABI and the corn transformation vector. To regenerate transgenic corn plants the transgenic callus resulting from transformation is placed on media to initiate shoot development in plantlets which are transferred to potting soil for initial growth in a growth chamber followed by a mist bench before transplanting to pots where plants are grown to maturity. The plants are self fertilized and seed is harvested for screening as seed, seedlings or progeny R2 plants or hybrids, e.g., for yield trials in the screens indicated above.
Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and enhanced seed composition.
Example 7
Soybean Plant Transformation
This example illustrates the production and identification of transgenic soybean cells in seed of transgenic soybean plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or enhanced seed compositions as compared to control plants. Transgenic soybean cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium -mediated transformation using the soybean transformation constructs disclosed in Example 5. Soybean transformation is effected using methods disclosed in U.S. Pat. No. 6,384,301 where soybean meristem explants are wounded then inoculated and co-cultured with the soybean transformation vector, then transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots.
The transformation is repeated for each of the protein encoding DNAs identified in Table 2.
Transgenic shoots producing roots are transferred to the greenhouse and potted in soil. Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and enhanced seed composition.
Example 8
Selection of Transgenic Plants with Enhanced Agronomic Trait(s)
This example illustrates identification of nuclei of the invention by screening derived plants and seeds for an enhanced trait identified below.
Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Populations of transgenic seed and plants prepared in Examples 6 and 7 are screened to identify those transgenic events providing transgenic plant cells with a nucleus having recombinant DNA imparting an enhanced trait. Each population is screened for enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and heat, increased level of oil and protein in seed using assays described below. Plant cell nuclei having recombinant DNA with each of the genes identified in Table 2 and the identified homologs are identified in plants and seeds with at least one of the enhanced traits.
A. Selection for Enhanced Nitrogen Use Efficiency
Transgenic corn plants with nuclei of the invention are planted in fields with three levels of nitrogen (N) fertilizer being applied, i.e. low level (0 pounds per acre N), medium level (80 pounds per acre N) and high level (180 pounds per acre N). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used as the N source and apply by broadcast boom and incorporate with a field cultivator with rear rolling basket in the same direction as intended crop rows. Although there is no N applied in the low level treatment, the soil should still be disturbed in the same fashion as the treated area. Transgenic plants and control plants can be grouped by genotype and construct with controls arranged randomly within genotype blocks. For improved statistical analysis each type of transgenic plant can be tested by 3 replications and across 4 locations. Nitrogen levels in the fields are analyzed before planting by collecting sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus (P), potassium (K), organic matter and pH to provide baseline values. P, K and micronutrients are applied based upon soil test recommendations.
Transgenic corn plants prepared in Example 6 and which exhibit a 2 to 5% yield increase as compared to control plants when grown in the high nitrogen field are selected as having nuclei of the invention. Transgenic corn plants which have at least the same or higher yield as compared to control plants when grown in the medium nitrogen field are selected as having nuclei of the invention. Transgenic corn plants having a nucleus with DNA identified in Table 3 as imparting nitrogen use efficiency (LN) and homologous DNA are selected from a nitrogen use efficiency screen as having a nucleus of this invention.
B. Selection for Increased Yield
Many transgenic plants of this invention exhibit increased yield as compared to a control plant. Increased yield can result from enhanced seed sink potential, i.e. the number and size of endosperm cells or kernels and/or enhanced sink strength, i.e. the rate of starch biosynthesis. Sink potential can be established very early during kernel development, as endosperm cell number and size are determined within the first few days after pollination.
Much of the increase in corn yield of the past several decades has resulted from an increase in planting density. During that period, corn yield has been increasing at a rate of 2.1 bushels/acre/year, but the planting density has increased at a rate of 250 plants/acre/year. A characteristic of modern hybrid corn is the ability of these varieties to be planted at high density. Many studies have shown that a higher than current planting density should result in more biomass production, but current germplasm does not perform. well at these higher densities. One approach to increasing yield is to increase harvest index (HI), the proportion of biomass that is allocated to the kernel compared to total biomass, in high density plantings.
Effective yield selection of enhanced yielding transgenic corn events uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control. A useful target for increased yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Selection methods can be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons to statistically distinguish yield improvement from natural environmental effects. Each of the transgenic corn plants and soybean plants with a nucleus of the invention prepared in Examples 6 and 7 are screened for yield enhancement. At least one event from each of the corn plants is selected as having at least between 3 and 5% increase in yield as compared to a control plant as having a nucleus of this invention.
C. Selection for Enhanced Water Use Efficiency (WUE)
The following is a high-throughput method for screening for water use efficiency in a greenhouse to identify the transgenic corn plants with a nucleus of this invention. This selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment. The hydration status of the shoot tissues following the drought is also measured. The plant heights are measured at three time points. The first is taken just prior to the onset drought when the plant is 11 days old, which is the shoot initial height (SIH). The plant height is also measured halfway throughout the drought/re-water regimen, on day 18 after planting, to give rise to the shoot mid-drought height (SMH). Upon the completion of the final drought cycle on day 26 after planting, the shoot portion of the plant is harvested and measured for a final height, which is the shoot wilt height (SWH) and also measured for shoot wilted biomass (SWM). The shoot is placed in water at 40 degree Celsius in the dark. Three days later, the shoot is weighted to give rise to the shoot turgid weight (STM). After drying in an oven for four days, the shoots are weighted for shoot dry biomass (SDM). The shoot average height (SAH) is the mean plant height across the 3 height measurements. The procedure described above can be adjusted for +/−˜one day for each step given the situation.
To correct for slight differences between plants, a size corrected growth value is derived from SIH and SWH. This is the Relative Growth Rate (RGR). Relative Growth Rate (RGR) is calculated for each shoot using the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)×100]. Relative water content (RWC) is a measurement of how much (%) of the plant was water at harvest. Water Content (RWC) is calculated for each shoot using the formula [RWC %=(SWM−SDM)/(STM−SDM)×100]. Fully watered corn plants of this age run around 98% RWC.
Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for water use efficiency. Transgenic plants having at least a 1% increase in RGR and RWC as compared to control plants are identified as having enhanced water used efficiency and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting drought tolerance enhancement (DS, HS, SS, and PEG) and homologous DNA are identified as showing increased water use efficiency as compared to control plants and are selected as having a nucleus of this invention.
D. Selection for Growth Under Cold Stress
Cold germination assay—Three sets of seeds are used for the assay. The first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention are expressed in the seed. The second seed set is nontransgenic, wild-type negative control made from the same genotype as the transgenic events. The third set consisted of two cold tolerant and one cold sensitive commercial check lines of corn. All seeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide, Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the demonstration.
Corn kernels are placed embryo side down on blotter paper within an individual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seeds from an event are placed into one cell of the germination tray. Each tray can hold 21 transgenic events and 3 replicates of wildtype (LH244SDms+LR59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7C for 24 days (no light) in a Convrion® growth chamber (Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Two hundred and fifty milliliters of deionized water are added to each germination tray. Germination counts are taken 10th, 11th, 12th, 13th, 14th, 17th, 19th, 21st, and 24th day after start date of the demonstration. Seeds are considered germinated if the emerged radicle size is 1 cm. From the germination counts germination index is calculated.
The germination index is calculated as per:
Germination index=(Σ([ T+ 1 −n i ]*[P i −P i−1 ]))/ T
where T is the total number of days for which the germination assay is performed. The number of days after planting is defined by n. “i” indicated the number of times the germination had been counted, including the current day. P is the percentage of seeds germinated during any given rating. Statistical differences are calculated between transgenic events and wild type control. After statistical analysis, the events that show a statistical significance at the p level of less than 0.1 relative to wild-type controls will advance to a secondary cold selection. The secondary cold screen is conducted in the same manner of the primary selection only increasing the number of repetitions to ten. Statistical analysis of the data from the secondary selection is conducted to identify the events that show a statistical significance at the p level of less than 0.05 relative to wild-type controls.
Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for water use efficiency. Transgenic plants having at least a 5% increase in germination index as compared to control plants are identified as having enhanced cold stress tolerance and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting cold tolerance enhancement (CK or CS) and homologous DNA are identified as showing increased cold stress tolerance as compared to control plants and are selected as having a nucleus of this invention.
E. Screens for Transgenic Plant Seeds with Increased Protein and/or Oil Levels
The following is a high-throughput selection method for identifying plant seeds with improvement in seed composition using the Infratec® 1200 series Grain Analyzer, which is a near-infrared transmittance spectrometer used to determine the composition of a bulk seed sample. Near infrared analysis is a non-destructive, high-throughput method that can analyze multiple traits in a single sample scan. An NIR calibration for the analytes of interest is used to predict the values of an unknown sample. The NIR spectrum is obtained for the sample and compared to the calibration using a complex chemometric software package that provides a predicted values as well as information on how well the sample fits in the calibration.
Infratec® Model 1221, 1225, or 1227 analyzer with transport module by Foss North America is used with cuvette, item # 1000-4033, Foss North America or for small samples with small cell cuvette, Foss standard cuvette modified by Leon Girard Co. Corn and soy check samples of varying composition maintained in check cell cuvettes are supplied by Leon Girard Co. NIT collection software is provided by Maximum Consulting Inc. Calculations are performed automatically by the software. Seed samples are received in packets or containers with barcode labels from the customer. The seed is poured into the cuvettes and analyzed as received.
TABLE 23 Typical sample(s): Whole grain corn and soybean seeds Analytical time to run Less than 0.75 min per sample method: Total elapsed time per 1.5 minute per sample run: Typical and minimum Corn typical: 50 cc; minimum 30 cc sample size: Soybean typical: 50 cc; minimum 5 cc Typical analytical range: Determined in part by the specific calibration. Corn - moisture 5-15%, oil 5-20%, protein 5-30%, starch 50-75%, and density 1.0-1.3%. Soybean - moisture 5-15%, oil 15-25%, and protein 35-50%.
Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for increased protein and oil in seed. Transgenic inbred corn and soybean plants having an increase of at least 1 percentage point in the total percent seed protein or at least 0.3 percentage point in total seed oil and transgenic hybrid corn plants having an increase of at least 0.4 percentage point in the total percent seed protein as compared to control plants are identified as having enhanced seed protein or enhanced seed oil and are selected as having a nucleus of this invention.

Example 9
Cotton Transgenic Plants with Enhanced Agronomic Traits
Cotton transformation is performed as generally described in WO0036911 and in U.S. Pat. No. 5,846,797. Transgenic cotton plants containing each of the recombinant DNA having a sequence of SEQ ID NO: 1 through SEQ ID NO: 114 are obtained by transforming with recombinant DNA from each of the genes identified in Table 1. Progeny transgenic plants are selected from a population of transgenic cotton events under specified growing conditions and are compared with control cotton plants. Control cotton plants are substantially the same cotton genotype but without the recombinant DNA, for example, either a parental cotton plant of the same genotype that was not transformed with the identical recombinant DNA or a negative isoline of the transformed plant. Additionally, a commercial cotton cultivar adapted to the geographical region and cultivation conditions, i.e. cotton variety ST474, cotton variety FM 958, and cotton variety Siokra L-23, are used to compare the relative performance of the transgenic cotton plants containing the recombinant DNA. The specified culture conditions are growing a first set of transgenic and control plants under “wet” conditions, i.e. irrigated in the range of 85 to 100 percent of evapotranspiration to provide leaf water potential of −14 to −18 bars, and growing a second set of transgenic and control plants under “dry” conditions, i.e. irrigated in the range of 40 to 60 percent of evapotranspiration to provide a leaf water potential of −21 to −25 bars. Pest control, such as weed and insect control is applied equally to both wet and dry treatments as needed. Data gathered during the trial includes weather records throughout the growing season including detailed records of rainfall; soil characterization information; any herbicide or insecticide applications; any gross agronomic differences observed such as leaf morphology, branching habit, leaf color, time to flowering, and fruiting pattern; plant height at various points during the trial; stand density; node and fruit number including node above white flower and node above crack boll measurements; and visual wilt scoring. Cotton boll samples are taken and analyzed for lint fraction and fiber quality. The cotton is harvested at the normal harvest timeframe for the trial area. Enhanced water use efficiency is indicated by increased yield, improved relative water content, enhanced leaf water potential, increased biomass, enhanced leaf extension rates, and improved fiber parameters.
The transgenic cotton plants of this invention are identified from among the transgenic cotton plants by agronomic trait screening as having increased yield and enhanced water use efficiency.
Example 10
Canola Plants with Enhanced Agrominic Traits
This example illustrates plant transformation useful in producing the transgenic canola plants of this invention and the production and identification of transgenic seed for transgenic canola having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
Tissues from in vitro grown canola seedlings are prepared and inoculated with a suspension of overnight grown Agrobacterium containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette. Following co-cultivation with Agrobacterium , the infected tissues are allowed to grow on selection to promote growth of transgenic shoots, followed by growth of roots from the transgenic shoots. The selected plantlets are then transferred to the greenhouse and potted in soil. Molecular characterizations are performed to confirm the presence of the gene of interest, and its expression in transgenic plants and progenies. Progeny transgenic plants are selected from a population of transgenic canola events under specified growing conditions and are compared with control canola plants. Control canola plants are substantially the same canola genotype but without the recombinant DNA, for example, either a parental canola plant of the same genotype that is not transformed with the identical recombinant DNA or a negative isoline of the transformed plant
Transgenic canola plant cells are transformed with recombinant DNA from each of the genes identified in Table 2. Transgenic progeny plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
Example 11
Monocot and Dicot Plant Transformation for the Suppression of Endogeneous Protein
This example illustrates monocot and dicot plant transformation to produce nuclei of this invention in cells of a transgenic plant by transformation where the recombinant DNA suppresses the expression of an endogenous protein identified in Table 24.
Corn callus and soybean tissue are transformed as describe in Examples 6 and 7 using recombinant DNA in the nucleus with DNA that transcribes to RNA that forms double-stranded RNA targeted to an endogenous gene with DNA encoding the protein. The genes for which the double-stranded RNAs are targeted are the native gene in corn and soybean that are homolog of the genes encoding the protein of Arabidopsis thaliana as identified in table 24.
Populations of transgenic plants prepared in Examples 6, 7 or 10 with DNA for suppressing a gene identified in Table 3 as providing an enhanced trait by gene suppression are screened to identify an event from those plants with a nucleus of the invention by selecting the trait identified in this specification.








TABLE 24



PEP SEQ

Construct


ID
Pfam module
ID
Traits











115
AP2
10177
LN


116
zf-C2H2
12155
CS
SS

118
F-box::Tub
11873
LN
PEG

154
Pex2_Pex12::zf-C3HC4
11113
CS

188
Myb_DNA-binding::Myb_DNA-
12325
LN


binding

200
bZIP_1
71228
CK