US20080295196A1 - Genes and uses for plant improvement - Google Patents

Genes and uses for plant improvement Download PDF

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Publication number
US20080295196A1
US20080295196A1 US12/001,025 US102507A US2008295196A1 US 20080295196 A1 US20080295196 A1 US 20080295196A1 US 102507 A US102507 A US 102507A US 2008295196 A1 US2008295196 A1 US 2008295196A1
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United States
Prior art keywords
protein
plants
sense
plant
seed
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Abandoned
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US12/001,025
Inventor
Mark S. Abad
Barry S. Goldman
Jaishree M. Chittoor-Vijayanath
Marie Coffin
Ronald C. Rich
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Monsanto Technology LLC
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Monsanto Technology LLC
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Priority to US12/001,025 priority Critical patent/US20080295196A1/en
Assigned to MONSANTO TECHNOLOGY LLC reassignment MONSANTO TECHNOLOGY LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ABAD, MARK, COFFIN, MARIE, CHITTOOR-VIJAYANATH, JAISHREE M., GOLDMAN, BARRY S., RICH, RONALD C.
Assigned to MONSANTO TECHNOLOGY LLC reassignment MONSANTO TECHNOLOGY LLC CORRECTIVE ASSIGNMENT TO CORRECT THE ASSIGNORS (SPECIFICALLY TO INCLUDE ASSIGNMENTS OF PREVIOUSLY MISSING INVENTORS FATEN SHAIKH AND DIANA WRAY) PREVIOUSLY RECORDED ON REEL 020758 FRAME 0752. ASSIGNOR(S) HEREBY CONFIRMS THE REMAINDER OF THE INFORMATION IN THE ORIGINAL ASSIGNMENT IS CORRECT. Assignors: SHAIKH, FATEN, WRAY, DIANA, ABAD, MARK S., COFFIN, MARIE, CHITTOOR-VIJAYANATH, JAISHREE M., GOLDMAN, BARRY S., RICH, RONALD C.
Assigned to MONSANTO TECHNOLOGY LLC reassignment MONSANTO TECHNOLOGY LLC CORRECTIVE ASSIGNMENT TO CORRECT THE NAMES OF THE INVENTORS (SPECIFICALLY BY INCLUDING THE NAME OF MISSING INVENTOR, JOSEPH C. MITCHELL). PREVIOUSLY RECORDED ON REEL 020758 FRAME 0752. ASSIGNOR(S) HEREBY CONFIRMS THE REMAINDER OF THE INFORMATION RECORDED ON THE ATTACHED ASSIGNMENT COVER PAGE, DATED 04/04/2008, IS CORRECT. Assignors: MITCHELL, JOSEPH C.
Publication of US20080295196A1 publication Critical patent/US20080295196A1/en
Priority to US13/372,542 priority patent/US20120167245A1/en
Priority to US13/914,701 priority patent/US9315822B2/en
Priority to US14/998,939 priority patent/US10093943B2/en
Priority to US16/350,019 priority patent/US20190153465A1/en
Abandoned legal-status Critical Current

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    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • G—PHYSICS
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Definitions

  • Folder hmmer-2.3.2 contains the source code and other associated file for implementing the HMMer software for Pfam analysis.
  • Folder 161pfamDir contains 161 profile Hidden Markov Models. Both folders were created on the disk on Dec. 4, 2007 having a total size of 15,534,080 bytes when measured in MS-WINDOWS® operating system.
  • transgenic plant cells, plants and seeds comprising recombinant DNA and methods of making and using such plant cells, plants and seeds
  • Transgenic plants with enhanced traits such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers.
  • traits such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers.
  • the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits.
  • the ability to develop transgenic plants with improved traits depends in part on the identification of useful recombinant DNA for production of transformed plants with improved properties, e.g. by actually selecting a transgenic plant from a screen for such improved property.
  • An object of this invention is to provide transgenic plant cell nuclei, plant cells, plants and seeds by screening transgenic crop plants for one of more enhanced agronomic traits where the nucleus in cells of the plant or seed has recombinant DNA that was identified as imparting an improved trait in a model plant, e.g. Arabidopsis thaliana .
  • the model plant may exhibit an improved trait that corresponds to an enhanced agronomic trait, e.g. cold stress tolerance, water deficit stress tolerance, low nitrogen stress tolerance and the like.
  • the model plant may exhibit an improved trait that is a surrogate to an enhanced agronomic trait, e.g.
  • a further object of the invention is to provide screening methods requiring routine experimentation by which such transgenic plant cell nuclei, cells, plants and seeds can be identified by making a reasonable number of transgenic events and engaging in screening identified in this specification and illustrated in the examples.
  • This invention provides plant cell nuclei with recombinant DNA that imparts enhanced agronomic traits in transgenic plants having the nuclei in their cells.
  • Recombinant DNA in this invention is provided in a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having at least one amino acid domain in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam domain names identified in Table 17.
  • plant cells which express a protein having amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 198 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 394 and homologs thereof listed in Table 2.
  • Amino acid sequences of homologs are SEQ ID NO: 395 through 19,938.
  • the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 1 by annotation to a related protein in Genbank and alternatively identified in Table 16 by identification of protein domain family.
  • transgenic plant cells and transgenic plants comprising a plurality of plant cells with such nuclei, progeny transgenic seed, embryo and transgenic pollen from such plants.
  • plant cell nuclei are selected from a population of transgenic plants regenerated from plant cells with a nucleus transformed with recombinant DNA by screening the transgenic plants in the population for an enhanced trait as compared to control plants that do not have the recombinant DNA in their nucleus, where the enhanced trait is enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced shade tolerance, enhanced tolerance to salt exposure, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil.
  • the recombinant DNA expresses a protein that imparts the enhanced trait; in other aspects of the invention the recombinant DNA expresses RNA for suppressing the level of an endogenous protein.
  • the nucleus of plant cells in plants, seeds, embryo and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type plant cell. Such tolerance is especially useful not only as an advantageous trait in such plants but is also useful in a selection step in the methods of the invention.
  • the agent of such herbicide is a glyphosate, dicamba, or glufosinate compound.
  • nuclei in cells of transgenic plants which are homozygous for the recombinant DNA and transgenic seed of the invention from corn, soybean, cotton, canola, alfalfa, wheat or rice plants.
  • the recombinant DNA in the nucleus is provided in plant cells derived from corn lines that are and maintain resistance to a virus such as the Mal de Rio Cuarto virus or a fungus such as the Puccina sorghi fungus or to both.
  • 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.
  • the recombinant DNA can express a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 17; in other aspects the recombinant DNA suppresses the level of such a protein.
  • the method comprises (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; (c) verifying that the recombinant DNA is stably integrated in said selected plants; (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO: 1-197; and (e) collecting seed from a selected plant.
  • the plants in the population further comprise 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.
  • the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, alfalfa, wheat or rice seed selected as having at least one of the enhanced traits described above.
  • Another aspect of the invention provides a method of producing hybrid corn seed comprising 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 further comprises 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 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.
  • Another aspect of the invention provides a method of selecting a plant comprising a nucleus of this invention in its plant cells by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue.
  • Another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a nucleus of this invention with unique recombinant DNA.
  • aspects of the invention relating to nucleus in plant cells having recombinant DNA for suppressing the expression of a protein are identified in Table 1 and Table 16. More specific aspects of the invention provide plant cells having recombinant DNA for suppressing the expression of a protein having the function in a plant of the protein with amino acid sequence of SEQ ID NO: 200, 201, 205, 207, 211 and 394 or the corresponding Pfam identified in Table 16, i.e. SNF5, LMBR1, TFIIS_M, TFIIS_C, Glyco_transf — 8, respectively.
  • Such suppression can be effected by any of a number of ways known in the art, e.g. anti-sense suppression, RNAi or mutation knockout and the like.
  • Another aspect of this invention relates to growing transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency.
  • this invention provides methods of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency.
  • methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop.
  • This invention also provides methods of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency.
  • methods comprise applying reduced amount of nitrogen input as compared to the conventional input during the production of a corn crop.
  • the various aspects of this invention are especially useful for transgenic plant cells in seeds and transgenic plants having any of the above-described enhanced traits in crop plants such as corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
  • crop plants such as corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
  • the invention also provides recombinant DNA constructs comprising the DNA useful in the nuclei in plant cells for imparting enhanced traits in plants having those cells.
  • FIG. 1 illustrates a consensus amino acid sequence of SEQ ID NO: 227 and its homologs.
  • FIGS. 2 , 3 and 4 illustrate plasmid maps.
  • SEQ ID NO: 1-197 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: 198-394 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequence 1-197;
  • SEQ ID NO: 395-19938 are amino acid sequences of homologous proteins
  • SEQ ID NO: 19939 is a consensus amino acid sequence.
  • SEQ ID NO: 19940 is a nucleotide sequence of a plasmid base vector useful for corn transformation.
  • SEQ ID NO: 19941 is a DNA sequence of a plasmid base vector useful for soybean or canola transformation.
  • SEQ ID NO: 19942 is a DNA sequence of a plasmid base vector useful for cotton transformation.
  • the nuclei of this invention are identified by screening transgenic plants for one or more traits including improved drought stress tolerance, improved heat stress tolerance, improved cold stress tolerance, improved high salinity stress tolerance, improved low nitrogen availability stress tolerance, improved shade stress tolerance, improved plant growth and development at the stages of seed imbibition through early vegetative phase, and improved plant growth and development at the stages of leaf development, flower production and seed maturity.
  • Gene refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequences involved in the regulation of expression.
  • gene refers at least to coding nucleotide sequence for a protein or a functional polypeptide fragment of a protein that imparts the trait.
  • gene refers to any part of the gene that can be a target for suppression.
  • Transgenic seed means a plant seed whose nucleus has been altered by the incorporation of recombinant DNA, e.g., by transformation as described herein.
  • the term “transgenic plant” is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains a nucleus with the recombinant DNA in its genome.
  • Recombinant DNA means a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous elements in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit.
  • Recombinant DNA may comprise 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.
  • a recombinant polynucleotide may exist outside of the cell, for example as a PCR fragment, or integrated into a genome, such as a plant genome.
  • Trait means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.
  • a “control plant” is a plant without trait-improving recombinant DNA in its nucleus.
  • a control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA.
  • a suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant herein.
  • a control plant may be a transgenic plant that comprises an empty vector or marker gene, but does not contain the recombinant DNA that produces the trait improvement.
  • a control plant may also be a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, the use of a limited number of control plants can cause a wide variation in the control dataset.
  • a “reference” is used.
  • 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, e.g., 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.
  • the trait enhancement can be measured quantitatively.
  • the trait improvement 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.
  • the trait enhancement is only measured qualitatively. It is known that there can be a natural variation in a trait.
  • 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 may 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 and high plant density.
  • 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, and juvenile traits.
  • 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.
  • transgenic plants that demonstrate desirable phenotypic properties that may or may not 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.
  • water deficit stress used herein preferably 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 stress conditions. Conditions that may result in water deficit stress include 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.
  • Nonrogen 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.
  • 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.
  • a low 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 may become exacerbated when the planting density exceeds the average prevailing density for a particular plant species.
  • the average prevailing densities per acre of a few examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre (Cheikh, e.g., (2003) U.S. Patent Application No. 20030101479).
  • “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.
  • 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 improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved 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 improved 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.
  • “Expression” means transcription of DNA to produce RNA.
  • the resulting RNA may be without limitation mRNA encoding a protein, antisense RNA, or a double-stranded RNA for use in RNAi technology.
  • Expression also refers to production of encoded protein from mRNA.
  • 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 comprise 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 may 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.
  • “antisense orientation” includes reference to a polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
  • operably linked refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • a “consensus sequence” refers to an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.
  • Homologous genes are genes which encode proteins with the same or similar biological function to the protein encoded by the second gene. Homologous genes may 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.
  • Percent identity refers to the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, e.g., 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 which 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.
  • “% identity to a consensus amino acid sequence” is 100 times the identity fraction in a window of alignment of an amino acid sequence of a test protein optimally aligned to consensus amino acid sequence of this invention.
  • Arabidopsis means plants of Arabidopsis thaliana.
  • Pfam refers to a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 18.0 (August 2005) contains alignments and models for 7973 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. Pfam is currently maintained and updated by a 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 Pfam 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.
  • profile HMMs Profile hidden Markov models
  • HMMER software and Pfam databases are version 18.0 and were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 198 through SEQ ID NO: 394. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 17 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.
  • Pfams for use in this invention are L51_S25_CI-B8, iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap, Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RmaAD, NAF, BolA, Pkinase, C2, FA_hydroxylase, p450, Complex1 — 30 kDa, Histone, DUF822, PEP-utilizers, PCI, ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE1, FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro2, PfkB, ORMDL, ADH_zinc_N, SWIM, Tr
  • the present invention provides recombinant DNA constructs comprising one or more polynucleotides disclosed herein for imparting one or more improved traits to transgenic plant when incorporated into the nucleus of the plant cells.
  • Such constructs also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the plant cells.
  • Other construct components may include additional regulatory elements, such as 5′ or 3′ untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides.
  • 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.
  • a polynucleotide of the present invention is operatively linked in a recombinant DNA construct to a promoter functional in a plant to provide for expression of the polynucleotide in the sense orientation such that a desired protein or polypeptide fragment of a protein is produced. Also provided are embodiments wherein a polynucleotide is operatively linked to a promoter functional in a plant to provide for expression of gene suppression RNA to suppress the level of an endogenous protein.
  • 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.
  • UTR 3′ untranslated DNA region
  • 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 may also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle.
  • a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle.
  • Table 1 provides a list of genes that provided recombinant DNA that was expressed in a model plant and identified from screening as imparting an improved trait.
  • 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.
  • 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.
  • the expression/suppression in the model plant exhibited an improved trait that corresponds to an enhanced agronomic trait, e.g. cold stress tolerance, water deficit stress tolerance, low nitrogen stress tolerance and the like.
  • the expression/suppression in the model plant exhibited an improved trait that is a surrogate to an enhance 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.
  • salinity stress tolerance being a surrogate to drought tolerance or improvement in plant growth and development being a surrogate to enhanced yield.
  • transgenic plant cell nuclei, cell, plant or seed of this invention can be identified by making a reasonable number of transgenic events and engaging in screening process identified in this specification and illustrated in the examples.
  • An understanding of Table 1 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.
  • 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:197, 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:197 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 may be naturally occurring, including DNA from homologous genes from the same or a different species, or may 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.
  • a DNA useful in the present invention may have any base sequence that has been changed from the sequences provided herein by substitution in accordance with degeneracy of the genetic code.
  • DNA is substantially identical to a reference DNA if, when the sequences of the polynucleotides are optimally aligned there is about 60% nucleotide equivalence; more preferably 70%; more preferably 80% equivalence; more preferably 85% equivalence; more preferably 90%; more preferably 95%; and/or more preferably 98% or 99% equivalence over a comparison window.
  • a comparison window is preferably at least 50-100 nucleotides, and more preferably is the entire length of the polynucleotide provided herein.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by algorithms; preferably 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 may be a full-length molecule or a portion of a longer molecule.
  • the window of comparison for determining polynucleotide identity of protein encoding sequences is the entire coding region.
  • Proteins useful for imparting improved traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins useful for generation of transgenic plants having improved traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 198 through SEQ ID NO: 394, as well as homologs of such proteins.
  • Homologs of the proteins useful in the invention are identified by comparison of the amino acid sequence of the protein to amino acid sequences of proteins from the same or different plant sources, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman.
  • a homolog is a protein from the same or a different organism that performs the same biological function as the polypeptide to which it is compared.
  • An orthologous relation between two organisms is not necessarily manifest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism phylogenetic separation, such as speciation. For a given protein, there may be no ortholog or more than one ortholog.
  • a local sequence alignment program e.g., BLAST
  • E-value Expectation value
  • BLAST BLAST
  • a reciprocal BLAST search is used in the present invention to filter hit sequences with significant E-values for ortholog identification.
  • the reciprocal BLAST 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 is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation.
  • homolog is used herein to describe proteins that are assumed to have functional similarity by inference from sequence base similarity.
  • the relationship of homologs with amino acid sequences of SEQ ID NO: 395 to SEQ ID NO: 19,938 to the proteins with amino acid sequences of SEQ ID NO: to 198 to SEQ ID NO: 394 are found in the listing of Table 2.
  • 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.
  • 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.
  • conserveed 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.
  • 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
  • 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 comprises 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.
  • Homologs of the trait-improving proteins provided herein will generally demonstrate significant sequence identity.
  • useful proteins also include those with higher identity, e.g., 90% to 99% identity.
  • Identity of protein homologs is determined by optimally 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: 198 through SEQ ID NO: 394.
  • Protein homologs include proteins with an amino acid sequence that has at least 90% identity to such a consensus amino acid sequence sequences.
  • 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.
  • NOS nopaline synthase
  • OCS octopine synthase
  • caulimovirus promoters such as the cauliflower mosaic virus or Figwort mosaic virus promoters.
  • CaMV35S cauliflower mosaic virus
  • the promoters can include multiple “enhancer sequences” to assist in elevating gene expression.
  • enhancers are known in the art.
  • the expression of the selected protein may 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.
  • 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.
  • enhancers examples 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.
  • the promoter element in the DNA construct 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 HVA22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea maize .
  • HSP17.5 heat shock protein 17.5 gene
  • HVA22 HVA22
  • Rab17 a cinnamic acid 4-hydroxylase
  • CA4H cinnamic acid 4-hydroxylase
  • 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).
  • 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
  • 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.
  • dsRNA double-stranded RNA
  • Suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function.
  • transformation with the T-DNA of Agrobacterium may be readily achieved and large numbers of transformants can be rapidly obtained.
  • 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 may be screened with polynucleotides encoding the polypeptide of interest to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.
  • 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.
  • the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the proteins disclosed herein.
  • Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
  • the methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera.
  • the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include using glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis ), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
  • a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
  • mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified antifungal protein, polypeptide or peptide.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible.
  • the use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol.
  • B cells B lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 ⁇ 10 7 to 2 ⁇ 10 8 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp. 75-83).
  • the immunized animal is a mouse
  • P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
  • NS-1 myeloma cell line also termed P3-NS-1-Ag-4-1
  • P3-NS-1-Ag-4-1 Another preferred murine myeloma cell
  • Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Spend virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al., 1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).
  • Fusion procedures usually produce viable hybrids at low frequencies, about 1 ⁇ 10 ⁇ 6 to 1 ⁇ 10 ⁇ 8 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azasenne blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
  • the cell lines may be exploited for mAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • transformation constructs will include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.
  • heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line.
  • it may 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.
  • site specific recombination systems exist which are known to function implants include 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 are preferably 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, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may 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.
  • 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.
  • 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 transgenic DNA construct into their genomes.
  • Preferred 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 may 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) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable 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 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.
  • Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay may 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 preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk 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 may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
  • Progeny may 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.
  • “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
  • Arabidopsis thaliana was transformed with a candidate recombinant DNA construct and screened for an improved trait.
  • Arabidopsis thaliana is used a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well-documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz, e.g., Methods in Arabidopsis Research e.g., (1992), World Scientific, New Jersey, New Jersey, in “Preface”).
  • a two-step screening process was employed which comprised 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 improved traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an improved trait.
  • Table 3 summarizes the improved traits that have been confirmed as provided by a recombinant DNA construct.
  • 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 improvement in a transgenic plant provided by the recombinant DNA.
  • the codes for improved traits are:
  • PEG which indicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen
  • SP which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein.
  • subtilis str. 168 283 73141 0 95 ref
  • Atroseptica SCRI1043 287 73120 8.00E ⁇ 82 100 ref
  • subtilis str. 168 311 73568 0 97 emb
  • CK PP PCC 7120 371 77155 0 97 emb
  • DS-Improvement 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 may find particular use in generating transgenic plants that are tolerant to the drought condition imposed during flowering time and in other stages of the plant life cycle.
  • 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-Improvement 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).
  • PEG polyethylene glycol
  • a PEG-induced osmotic stress tolerance screen is a useful surrogate for drought tolerance 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-Improvement 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.
  • trait-improving recombinant DNA identified in a high salinity stress tolerance screen can also provide transgenic crops with improved 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-Improvement 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 may also impart improved 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-Improvement of tolerance to cold stress Low temperature may immediately result in mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential.
  • two screening conditions i.e., cold shock tolerance screen (CK) and cold germination tolerance screen (CS)
  • CK cold shock tolerance screen
  • CS 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.
  • 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.
  • 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.
  • PEP SEQ ID NO: 231 can be used to improve both salt stress tolerance and cold stress tolerance in plants.
  • plants transformed with PEP SEQ ID NO: 233 can resist heat stress, salt stress and cold stress.
  • the stress tolerance conferring genes provided by the present invention may be used in combinations to generate transgenic plants that can resist multiple stress conditions.
  • 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.
  • 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 may also be used to generate transgenic plants that are more resistant to various stress conditions due to improved 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: 268 which can improve the plant early growth and development, and impart heat and cold tolerance to plants.
  • PEP SEQ ID NO: 268 which can improve the plant early growth and development, and impart heat and cold tolerance to plants.
  • 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.
  • “Late growth and development” used herein encompasses the stages of leaf development, flower production, and seed maturity.
  • 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.
  • 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.
  • 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.
  • embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen can grow better and/or have improved development during leaf development and seed maturation providing a higher yield potential as compared to control plants.
  • LL-Improvement 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.
  • 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.
  • 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 may be suitable for a higher density planting, thereby resulting increased yield per unit area.
  • 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.
  • the transgenic plants provided by the present invention with enhanced nitrogen use efficiency may also have altered amino acid or protein compositions, increased yield and/or better seed quality.
  • the transgenic plants of the present invention may 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 may 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.
  • the present invention also encompasses transgenic plants with stacked engineered traits, e.g., a crop having an improved phenotype resulting from expression of a trait-improving recombinant DNA, in combination with herbicide and/or pest resistance traits.
  • 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.
  • glyphosate herbicides glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides.
  • 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: 197, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO: 198 through SEQ ID NO: 394.
  • the present invention provides the protein sequences of identified homologs for a sequence listed as SEQ ID NO: 395 through SEQ ID NO: 19938.
  • 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 improved traits provided by the present invention are not limited to any particular plant species.
  • the plants according to the present invention may be of any plant species, i.e., may be monocotyledonous or dicotyledonous.
  • 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 improved traits include, without limitation, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
  • the present invention contemplates to use an orthologous gene in generating the transgenic plants with similarly improved traits as the transgenic Arabidopsis counterpart.
  • Improved physiological properties in transgenic plants of the present invention may be confirmed in responses to stress conditions, for example in assays using imposed stress conditions to detect improved responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions.
  • Biomass measures may be made on greenhouse or field grown plants and may 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 may 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 may 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.
  • trait characteristics of harvested grain may 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.
  • hybrid yield in transgenic corn plants expressing genes of the present invention it may 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.
  • 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.
  • 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 ⁇ mol/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.
  • 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.
  • 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.
  • p ⁇ 0.05 and delta or risk score mean >0 the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance)
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 226, 237, 242, or 363 showed improved drought tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.
  • 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 1 ⁇ 2 ⁇ MS salts, 1% phytagel, with 10 ⁇ g/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 ⁇ mol/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.
  • the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 307, 313, 327, 330, 349, 366, or 387 showed improved heat stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.
  • 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 experiment 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.
  • the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 242, 255, 307, 313, 327, 330, 349, 366, or 387 showed improved salt stress tolerance evidenced by the second criteria as illustrated in Example 1L.
  • 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, 1 ⁇ 2 ⁇ MS salts, 1% phytagel, and 10 ⁇ g/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.
  • Table 7 A list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants illustrated in Table 7.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 209, 245, 260, 274, 282, 310, 321, 325, 336, 372, 376, 378, 380, or 382 showed improved osmotic stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.
  • 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.
  • p ⁇ 0.05 and delta or risk score mean >0 the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance)
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 255, 272, or 330 showed improved cold stress tolerance evidenced by the second criterial as illustrated in Example 1L.
  • 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 experiment were grown at 8° C. Seeds were first surface disinfested using chlorine gas and then seeded on assay plates containing an aqueous solution of 1 ⁇ 2 ⁇ Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% PhytagelTM (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.
  • 1 ⁇ 2 ⁇ Gamborg's B/5 Basal Salt Mixture Sigma/Aldrich Corp., St. Louis, Mo., USA G
  • 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.
  • the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 347 showed improved cold stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.
  • 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 1 ⁇ 2MS medium. Seeds were sown on 1 ⁇ 2 ⁇ 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.
  • seeding weight and “leaf angle”
  • p ⁇ 0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p ⁇ 0.2.
  • transgenic plants For “petiole length”, if p ⁇ 0.05 and delta ⁇ 0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2 and delta ⁇ 0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 246, 295, 303, 325, or 375 showed enhanced shade tolerance by the second criteria as illustrated in Example 1L and 1M.
  • 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.
  • 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.
  • the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 252, 309, or 310 showed improved early plant growth and development evidenced by the second criteria as illustrated in Example 1L and 1M.
  • 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 21 ⁇ 2′′ 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 ⁇ moles/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.
  • 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.
  • the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • 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.
  • the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p ⁇ 0.2. For root length, if p ⁇ 0.05, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p ⁇ 0.2, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 198 or 327 showed improved tolerance to low nitrogen condition evidenced by the second criteria as illustrated in Example 1L and 1M.
  • 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).
  • 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.
  • 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).
  • 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 events perform 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.
  • p ⁇ 0.05 and risk score mean >0 the transgenic plants from this event showed statistically significant trait enhancement as compared to the reference.
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • p ⁇ 0.05 and delta mean >0 the transgenic plants from this event showed statistically significant trait improvement as compared to the reference.
  • 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.
  • 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.
  • NCBI National Center for Biotechnology Information
  • 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: 198 through SEQ ID NO: 394 using “blastp” with E-value cutoff of 1e ⁇ 8.
  • sequences of SEQ ID NO: 198 through SEQ ID NO: 394 using “blastp” with E-value cutoff of 1e ⁇ 8.
  • Up to 1000 top hits were kept, and separated by organism names.
  • 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: 198 through SEQ ID NO: 394 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.
  • ClustalW program is selected for multiple sequence alignments of an amino acid sequence of SEQ ID NO: 198 and its homologs, through SEQ ID NO: 394 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: 227 and its 25 homologs were derived according to the procedure described above and is displayed in FIG. 1 .
  • This example illustrates the identification of amino acid domain 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 protein families for the proteins of SEQ ID NO: 198 through 394 are shown in Table 16.
  • 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: 207 is characterized by two Pfam domains, i.e.
  • TFIIS_M and “TFIIS_C”. See also the protein with amino acids of SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”.
  • SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”.
  • SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”.
  • SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”.
  • SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”.
  • SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”.
  • SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”.
  • SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”.
  • SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”.
  • 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 1 and the DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the base vector.
  • Elements of an exemplary common expression vector, pMON93093 are illustrated in Table 18.
  • the exemplary base vector which is especially useful for corn transformation is illustrated in FIG. 2 and assembled using technology known in the art.
  • the DNA of interest are inserted in a expression vector at the insertion site between the intron1 of rice act 1 gene and the termination sequence of PinII 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.
  • Agro B-AGRtu.left border Agro left border 6168-6609 transformation sequence, essential for transfer of T-DNA.
  • Maintenance OR-Ec.oriV-RK2 The vegetative origin of 6696-7092 in E. coli replication from plasmid RK2.
  • OR-Ec.ori-ColE1 The minimal origin of 9220-9808 replication from the E. coli plasmid ColE1.
  • Plasmids for use in transformation of soybean are also prepared. Elements of an exemplary common expression vector plasmid pMON82053 are shown in Table 19 below.
  • This exemplary soybean or canola transformation base vector illustrated in FIG. 3 is assembled using the technology known in the art. Recombinant DNA of interest, i.e. each DNA identified in Table 1 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.
  • 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; Agro B-AGRtu.right Agro right border sequence, essential 1033-1389 transformation border for transfer of T-DNA.
  • OR-Ec.oriV-RK2 The vegetative origin of replication 5661-6057 in E. coli from plasmid RK2.
  • OR-Ec.ori-ColE1 The minimal origin of replication 2945-3533 from the E. coli plasmid ColE1.
  • Plasmids for use in transformation of cotton are also prepared. Elements of an exemplary common expression vector plasmid pMON99053 are shown in Table 20 below and FIG. 4 . 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 1 is amplified by PCR prior to insertion into the insertion site within the gene of interest expression cassette of one of the base.
  • OR-Ec.oriV-RK2 The vegetative origin of 2739-3135 E. coli replication from plasmid RK2.
  • OR-Ec.ori-ColE1 The minimal origin of 5263-5851 replication from the E. coli plasmid ColE1.
  • Transgenic corn cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium -mediated transformation using the corn transformation vectors pMON93093 as disclosed in Example 6.
  • 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.
  • 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.
  • 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 improved seed composition.
  • 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 vectors pMON82053 disclosed in Example 7. 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.
  • 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 improved seed composition.
  • 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 overnight-grown Agrobacterium cells 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 characterization 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 1.
  • 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 as reported in Example 9.
  • This example illustrates identification of nuclei of the invention by screening derived plants and seeds for an enhanced trait identified below.
  • 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 1 and the identified homologs are identified in plants and seeds with at least one of the enhanced traits.
  • 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.
  • transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved 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.
  • 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 improved 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 may 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. It is to plant multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, for example 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges.
  • Transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field.
  • a pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events.
  • a useful planting density is about 30,000 plants/acre.
  • High planting density is greater than 30,000 plants/acre, preferably about 40,000 plants/acre, more preferably about 42,000 plants/acre, most preferably about 45,000 plants/acre.
  • 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 and soybean 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.
  • 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 height are measured at three time points.
  • SIH shoot initial height
  • SWH shoot wilt height
  • SWM shoot wilted biomass
  • STM shoot turgid weight
  • SDM shoot dry biomass
  • 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 improvement (DS) 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.
  • 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 experiment.
  • Captan MAESTRO® 80DF Fungicide
  • 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+LH59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7 C 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.
  • Convrion® growth chamber Convrion® growth chamber
  • Germination counts are taken 10th, 11th, 12th, 13th, 14th, 17th, 19th, 21st, and 24th day after start date of the experiment. 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 ( ⁇ ([ T+ 1 ⁇ n i ]*[P i ⁇ P i ⁇ 1 ]))/ T
  • 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 improvement (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.
  • 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.
  • 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 run: 1.5 minute per sample Typical and minimum sample Corn typical: 50 cc; minimum 30 cc 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.
  • 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: 197 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.
  • 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.
  • 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.
  • 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 by Pfam, SNF5, LMBR1, TFIIS_M, TFIIS_C, or Glyco_transf — 8.
  • 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 with an amino acid sequence of SEQ ID NO:200, 201, 205, 207, 211, and 394.

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Abstract

Transgenic seed for crops with improved 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 improved 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.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application claims benefit under 35USC § 119(e) of U.S. provisional application Ser. No. 60/873,247, filed Dec. 6, 2006, herein incorporated by reference.
    • 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(53949)B_seqListing.txt, which is 67,317,760 bytes (measured in MS-WINDOWS) and was created on Dec. 4, 2007, are incorporated herein by reference in their entirety.
    • INCORPORATION OF COMPUTER PROGRAM LISTING
  • A Computer Program Listing with folders “hmmer-2.3.2” and “161pfamDir” 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 161pfamDir contains 161 profile Hidden Markov Models. Both folders were created on the disk on Dec. 4, 2007 having a total size of 15,534,080 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
    • BACKGROUND OF THE INVENTION
  • Transgenic plants with enhanced traits such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. The ability to develop transgenic plants with improved traits depends in part on the identification of useful recombinant DNA for production of transformed plants with improved properties, e.g. by actually selecting a transgenic plant from a screen for such improved property. An object of this invention is to provide transgenic plant cell nuclei, plant cells, plants and seeds by screening transgenic crop plants for one of more enhanced agronomic traits where the nucleus in cells of the plant or seed has recombinant DNA that was identified as imparting an improved trait in a model plant, e.g. Arabidopsis thaliana. In some cases the model plant may exhibit an improved 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 model plant may exhibit an improved 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. A further object of the invention is to provide screening methods requiring routine experimentation by which such transgenic plant cell nuclei, cells, plants and seeds can be identified by making a reasonable number of transgenic events and engaging in screening identified in this specification and illustrated in the examples.
    • 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. Recombinant DNA in this invention is provided in a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having at least one amino acid domain in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam domain names identified in Table 17. In more specific embodiments of the invention plant cells are provided which express a protein having amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 198 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 394 and homologs thereof listed in Table 2. Amino acid sequences of homologs are SEQ ID NO: 395 through 19,938. In even more specific embodiments of the invention the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 1 by annotation to a related protein in Genbank and alternatively identified in Table 16 by identification of protein domain family.
  • Other aspects of the invention are specifically directed to transgenic plant cells, and transgenic plants comprising a plurality of plant cells with such nuclei, progeny transgenic seed, embryo and transgenic pollen from such plants. Such plant cell nuclei are selected from a population of transgenic plants regenerated from plant cells with a nucleus transformed with recombinant DNA by screening the transgenic plants in the population for an enhanced trait as compared to control plants that do not have the recombinant DNA in their nucleus, where the enhanced trait is enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced shade tolerance, enhanced tolerance to salt exposure, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil. In some aspects of the invention the recombinant DNA expresses a protein that imparts the enhanced trait; in other aspects of the invention the recombinant DNA expresses RNA for suppressing the level of an endogenous protein. In yet another aspect of the invention the nucleus of plant cells in plants, seeds, embryo and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type plant cell. Such tolerance is especially useful not only as an advantageous trait in such plants but is also useful in a selection step in the methods of the invention. In aspects of the invention the agent of such herbicide is a glyphosate, dicamba, or glufosinate compound.
  • Yet other aspects of the invention provide nuclei in cells of transgenic plants which are homozygous for the recombinant DNA and transgenic seed of the invention from corn, soybean, cotton, canola, alfalfa, wheat or rice plants.
  • In other embodiments for practice of various aspects of the invention in Argentina the recombinant DNA in the nucleus is provided in plant cells derived from corn lines that are and maintain resistance to a virus such as the Mal de Rio Cuarto virus or a fungus such as the Puccina sorghi fungus or to both.
  • 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. In some aspects of the invention the recombinant DNA can express a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 17; in other aspects the recombinant DNA suppresses the level of such a protein. More specifically the method comprises (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; (c) verifying that the recombinant DNA is stably integrated in said selected plants; (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO: 1-197; and (e) collecting seed from a selected plant. In one aspect of the invention the plants in the population further comprise 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 are especially useful for manufacturing corn, soybean, cotton, alfalfa, wheat or rice seed selected as having at least one of the enhanced traits described above.
  • Another aspect of the invention provides a method of producing hybrid corn seed comprising 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 further comprises 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 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.
  • Another aspect of the invention provides a method of selecting a plant comprising a nucleus of this invention in its plant cells by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue. Another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a nucleus of this invention with unique recombinant DNA.
  • Aspects of the invention relating to nucleus in plant cells having recombinant DNA for suppressing the expression of a protein are identified in Table 1 and Table 16. More specific aspects of the invention provide plant cells having recombinant DNA for suppressing the expression of a protein having the function in a plant of the protein with amino acid sequence of SEQ ID NO: 200, 201, 205, 207, 211 and 394 or the corresponding Pfam identified in Table 16, i.e. SNF5, LMBR1, TFIIS_M, TFIIS_C, Glyco_transf—8, respectively. Such suppression can be effected by any of a number of ways known in the art, e.g. anti-sense suppression, RNAi or mutation knockout and the like.
  • Another aspect of this invention relates to growing transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency. For instance, this invention provides methods of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency. Alternatively methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop. This invention also provides methods of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency. Alternatively methods comprise applying reduced amount of nitrogen input as compared to the conventional input during the production of a corn crop.
  • The various aspects of this invention are especially useful for transgenic plant cells in seeds and transgenic plants having any of the above-described enhanced traits in crop plants such as corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
  • The invention also provides recombinant DNA constructs comprising the DNA useful in the nuclei in plant cells for imparting enhanced traits in plants having those cells.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 illustrates a consensus amino acid sequence of SEQ ID NO: 227 and its homologs.
  • FIGS. 2, 3 and 4 illustrate plasmid maps.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • In the attached sequence listing:
  • SEQ ID NO: 1-197 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: 198-394 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequence 1-197;
  • SEQ ID NO: 395-19938 are amino acid sequences of homologous proteins;
  • SEQ ID NO: 19939 is a consensus amino acid sequence.
  • SEQ ID NO: 19940 is a nucleotide sequence of a plasmid base vector useful for corn transformation; and
  • SEQ ID NO: 19941 is a DNA sequence of a plasmid base vector useful for soybean or canola transformation.
  • SEQ ID NO: 19942 is a DNA sequence of a plasmid base vector useful for cotton transformation.
  • The nuclei of this invention are identified by screening transgenic plants for one or more traits including improved drought stress tolerance, improved heat stress tolerance, improved cold stress tolerance, improved high salinity stress tolerance, improved low nitrogen availability stress tolerance, improved shade stress tolerance, improved plant growth and development at the stages of seed imbibition through early vegetative phase, and improved plant growth and development at the stages of leaf development, flower production and seed maturity.
  • “Gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequences involved in the regulation of expression. In aspects of the invention where an improved trait is provided by expression of a protein, “gene” refers at least to coding nucleotide sequence for a protein or a functional polypeptide fragment of a protein that imparts the trait. In aspects of the invention where an improved trait is provided by suppression of expression of an endogenous protein, “gene” refers to any part of the gene that can be a target for suppression.
  • “Transgenic seed” means a plant seed whose nucleus has been altered by the incorporation of recombinant DNA, e.g., by transformation as described herein. The term “transgenic plant” is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains a nucleus with the recombinant DNA in its genome.
  • “Recombinant DNA” means a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous elements in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit. Recombinant DNA may comprise 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. A recombinant polynucleotide may exist outside of the cell, for example as a PCR fragment, or integrated into a genome, such as a plant genome.
  • “Trait” means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.
  • A “control plant” is a plant without trait-improving recombinant DNA in its nucleus. A control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA. A suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant herein. Alternatively, a control plant may be a transgenic plant that comprises an empty vector or marker gene, but does not contain the recombinant DNA that produces the trait improvement. A control plant may also be a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, 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, e.g., 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 improvement 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 may 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 and high plant density.
  • 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, and juvenile traits. 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 may or may not 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 preferably 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 stress conditions. Conditions that may result in water deficit stress include 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 low 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 may become exacerbated when the planting density exceeds the average prevailing density for a particular plant species. The average prevailing densities per acre of a few examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre (Cheikh, e.g., (2003) U.S. Patent Application No. 20030101479).
  • “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 improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved 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 improved 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.
  • “Expression” means transcription of DNA to produce RNA. The resulting RNA may be without limitation mRNA encoding a protein, antisense RNA, or a double-stranded RNA for use in RNAi technology. Expression also refers to production of encoded protein from mRNA.
  • 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 comprise 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 may 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, “antisense orientation” includes reference to a polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
  • As used herein, “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • A “consensus sequence” refers to an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.
  • Homologous genes are genes which encode proteins with the same or similar biological function to the protein encoded by the second gene. Homologous genes may 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.
  • Percent identity refers to the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, e.g., 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 which 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. “% identity to a consensus amino acid sequence” is 100 times the identity fraction in a window of alignment of an amino acid sequence of a test protein optimally aligned to consensus amino acid sequence of this invention.
  • “Arabidopsis” means plants of Arabidopsis thaliana.
  • “Pfam” refers to a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 18.0 (August 2005) contains alignments and models for 7973 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. Pfam is currently maintained and updated by a 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 Pfam 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. Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins in the same protein family are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Model which characterizes the Pfam domain using HMMER software, a current version of which is provided in the appended computer listing. Candidate proteins meeting the gathering cutoff for the alignment of a particular Pfam 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 in a common Pfam for recombinant DNA in the plant cells of this invention are also included in the appended computer listing. The HMMER software and Pfam databases are version 18.0 and were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 198 through SEQ ID NO: 394. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 17 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 Pfams for use in this invention, as more specifically disclosed below, are L51_S25_CI-B8, iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap, Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RmaAD, NAF, BolA, Pkinase, C2, FA_hydroxylase, p450, Complex1—30 kDa, Histone, DUF822, PEP-utilizers, PCI, ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE1, FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro2, PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5, Ceramidase_alk, Ribonuclease_T2, Complex1—49 kDa, Gp_dh_C, Aldo_ket_red, zf-AN1, TFIIS_C, MFS—1, Thioredoxin, DUF1005, LEA—3, Sterol_MT_C, Gp_dh_N, TFIIS_M, PAN—2, BPL_C, DUF26, Aa_trans, ACT, ADH_N, NAD_binding—1, Auxin_inducible, B_lectin, Anti-silence, Response_reg, 14-3-3, LRRNT—2, GDC-P, zf-CCHC, NPH3, TPR—1, TFIIA, DHBP_synthase, UQ_con, TPR—2, TPT, F-box, adh_short, Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv, TIM, NDK, Pantoate_transf, Allene_ox_cyc, Cyclin_N, Methyltransf—1, CBM—20, Methyltransf—12, Rhodanese, Glycolytic, Actin, Usp, eIF-4B, Glyco_transf—8, BURP, Alpha-amylase, F420_oxidored, EGF_CA, Kelch—1, PGAM, Aminotran—1—2, Kelch—2, UPF0261, CoA_binding, DUF868, Peptidase_S10, Lung—7-TM_R, Oleosin, Sad1_UNC, Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase, Subtilisin_N, SAM—1, DUF298, ESCRT-III, DNA_pol_E_B, Aminotran—3, NAD_Gly3P_dh_N, Gln-synt_N, MMR_HSR1, DUF588, zf-CCCH, DnaJ, Pkinase_Tyr, Cupin—2, LRR—1, Cupin—3, zf-CSL, FAR1, HD, FH2, APC8, PTR2, MannoseP_isomer, Rib—5-P_isom_A, DUF1336, Phosphorylase, DUF1191, Asp, Mit_rib_S27, PAP_fibrillin, DUF1195, Aldedh, zf-C3HC4, PPR, PK, PurA, RMMBL, HTH—11, Tim17, and PBD.
    • Recombinant DNA Constructs
  • The present invention provides recombinant DNA constructs comprising one or more polynucleotides disclosed herein for imparting one or more improved traits to transgenic plant when incorporated into the nucleus of the plant cells. Such constructs also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the plant cells. Other construct components may 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.
  • In a preferred embodiment, a polynucleotide of the present invention is operatively linked in a recombinant DNA construct to a promoter functional in a plant to provide for expression of the polynucleotide in the sense orientation such that a desired protein or polypeptide fragment of a protein is produced. Also provided are embodiments wherein a polynucleotide is operatively linked to a promoter functional in a plant to provide for expression of gene suppression RNA to suppress the level of an endogenous protein.
  • 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 may 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 1 provides a list of genes that provided recombinant DNA that was expressed in a model plant and identified from screening as imparting an improved 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 improved 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 improved trait that is a surrogate to an enhance 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 improved 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. However, by with routine experimentation a transgenic plant cell nuclei, cell, plant or seed of this invention can be identified by making a reasonable number of transgenic events and engaging in screening process identified in this specification and illustrated in the examples. An understanding of Table 1 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 1
    NUC PEP
    Seq ID SEQ ID Construct
    No. No Gene ID ID Orientation
    1 198 CGPG106 11029 SENSE
    2 199 CGPG1133 12223 SENSE
    3 200 CGPG117 10422 ANTI-SENSE
    4 201 CGPG1226 13485 ANTI-SENSE
    5 202 CGPG1288 13235 SENSE
    6 203 CGPG1301 13411 SENSE
    7 204 CGPG1458 73944 SENSE
    8 205 CGPG1542 13846 ANTI-SENSE
    9 206 CGPG170 12602 SENSE
    10 207 CGPG1828 74065 ANTI-SENSE
    11 208 CGPG2206 72783 SENSE
    12 209 CGPG2217 17210 SENSE
    13 210 CGPG2292 72724 SENSE
    14 211 CGPG2457 17805 ANTI-SENSE
    15 212 CGPG2499 16610 SENSE
    16 213 CGPG2653 76602 SENSE
    17 214 CGPG2813 18456 SENSE
    18 215 CGPG3002 18414 SENSE
    19 216 CGPG3154 71538 SENSE
    20 217 CGPG3235 76532 SENSE
    21 218 CGPG3274 18231 SENSE
    22 219 CGPG3275 18232 SENSE
    23 220 CGPG3363 18256 SENSE
    24 221 CGPG3367 18258 SENSE
    25 222 CGPG3375 19193 SENSE
    26 223 CGPG3528 71301 SENSE
    27 224 CGPG3534 18354 SENSE
    28 225 CGPG3638 77334 SENSE
    29 226 CGPG3918 19767 SENSE
    30 227 CGPG3920 19774 SENSE
    31 228 CGPG3962 70992 SENSE
    32 229 CGPG3972 19956 SENSE
    33 230 CGPG3990 70948 SENSE
    34 231 CGPG3994 70201 SENSE
    35 232 CGPG4026 19973 SENSE
    36 233 CGPG4048 70987 SENSE
    37 234 CGPG4052 70950 SENSE
    38 235 CGPG4057 70962 SENSE
    39 236 CGPG4058 70915 SENSE
    40 237 CGPG4069 19947 SENSE
    41 238 CGPG4087 70969 SENSE
    42 239 CGPG4088 70985 SENSE
    43 240 CGPG4102 70971 SENSE
    44 241 CGPG4121 70963 SENSE
    45 242 CGPG4122 70994 SENSE
    46 243 CGPG4140 70956 SENSE
    47 244 CGPG4154 70995 SENSE
    48 245 CGPG4311 73306 SENSE
    49 246 CGPG4363 70657 SENSE
    50 247 CGPG4369 70660 SENSE
    51 248 CGPG442 74536 SENSE
    52 249 CGPG4454 71328 SENSE
    53 250 CGPG4456 71329 SENSE
    54 251 CGPG4473 70755 SENSE
    55 252 CGPG4588 70684 SENSE
    56 253 CGPG4765 73330 SENSE
    57 254 CGPG4788 76202 SENSE
    58 255 CGPG4912 72807 SENSE
    59 256 CGPG4926 72811 SENSE
    60 257 CGPG4967 73235 SENSE
    61 258 CGPG4977 72813 SENSE
    62 259 CGPG5001 72825 SENSE
    63 260 CGPG5025 73628 SENSE
    64 261 CGPG5041 76105 SENSE
    65 262 CGPG5116 73242 SENSE
    66 263 CGPG5144 74217 SENSE
    67 264 CGPG5171 73735 SENSE
    68 265 CGPG5194 73256 SENSE
    69 266 CGPG5200 73260 SENSE
    70 267 CGPG5210 75822 SENSE
    71 268 CGPG5221 72001 SENSE
    72 269 CGPG5269 72056 SENSE
    73 270 CGPG5404 77308 SENSE
    74 271 CGPG5432 73766 SENSE
    75 272 CGPG5518 72774 SENSE
    76 273 CGPG5535 72788 SENSE
    77 274 CGPG5540 72753 SENSE
    78 275 CGPG5568 72709 SENSE
    79 276 CGPG5577 73954 SENSE
    80 277 CGPG5587 73137 SENSE
    81 278 CGPG5594 73161 SENSE
    82 279 CGPG5633 73057 SENSE
    83 280 CGPG5640 73127 SENSE
    84 281 CGPG5646 73033 SENSE
    85 282 CGPG5656 73105 SENSE
    86 283 CGPG5659 73141 SENSE
    87 284 CGPG5661 73165 SENSE
    88 285 CGPG5684 73155 SENSE
    89 286 CGPG5694 73026 SENSE
    90 287 CGPG5704 73120 SENSE
    91 288 CGPG5714 73133 SENSE
    92 289 CGPG5721 73134 SENSE
    93 290 CGPG5728 73123 SENSE
    94 291 CGPG5757 73981 SENSE
    95 292 CGPG5764 73136 SENSE
    96 293 CGPG5783 73172 SENSE
    97 294 CGPG5791 73020 SENSE
    98 295 CGPG5799 72946 SENSE
    99 296 CGPG5856 74746 SENSE
    100 297 CGPG5927 77312 SENSE
    101 298 CGPG5941 75237 SENSE
    102 299 CGPG5957 75240 SENSE
    103 300 CGPG5967 74349 SENSE
    104 301 CGPG6040 76422 SENSE
    105 302 CGPG607 70812 SENSE
    106 303 CGPG6178 77322 SENSE
    107 304 CGPG6185 74662 SENSE
    108 305 CGPG6306 76527 SENSE
    109 306 CGPG6318 77020 SENSE
    110 307 CGPG6326 77609 SENSE
    111 308 CGPG6370 73485 SENSE
    112 309 CGPG6429 73433 SENSE
    113 310 CGPG6440 73411 SENSE
    114 311 CGPG6516 73568 SENSE
    115 312 CGPG6653 74688 SENSE
    116 313 CGPG6712 74420 SENSE
    117 314 CGPG6737 74435 SENSE
    118 315 CGPG6747 74460 SENSE
    119 316 CGPG6796 74566 SENSE
    120 317 CGPG6805 77610 SENSE
    121 318 CGPG6810 77618 SENSE
    122 319 CGPG6952 77517 SENSE
    123 320 CGPG6953 77518 SENSE
    124 321 CGPG7121 76460 SENSE
    125 322 CGPG7163 77069 SENSE
    126 323 CGPG7168 76161 SENSE
    127 324 CGPG7206 76171 SENSE
    128 325 CGPG7225 76178 SENSE
    129 326 CGPG7267 76467 SENSE
    130 327 CGPG7272 77536 SENSE
    131 328 CGPG7281 76576 SENSE
    132 329 CGPG7308 74862 SENSE
    133 330 CGPG7316 74863 SENSE
    134 331 CGPG7371 74858 SENSE
    135 332 CGPG7457 74933 SENSE
    136 333 CGPG7520 75379 SENSE
    137 334 CGPG7529 77816 SENSE
    138 335 CGPG7636 75434 SENSE
    139 336 CGPG7737 77821 SENSE
    140 337 CGPG7767 75685 SENSE
    141 338 CGPG7804 75654 SENSE
    142 339 CGPG7823 75692 SENSE
    143 340 CGPG7828 75657 SENSE
    144 341 CGPG7833 75622 SENSE
    145 342 CGPG7933 77549 SENSE
    146 343 CGPG7986 77917 SENSE
    147 344 CGPG8012 77568 SENSE
    148 345 CGPG8015 77570 SENSE
    149 346 CGPG8055 77338 SENSE
    150 347 CGPG8062 77580 SENSE
    151 348 CGPG8082 77928 SENSE
    152 349 CGPG8083 77349 SENSE
    153 350 CGPG8106 77357 SENSE
    154 351 CGPG8107 77587 SENSE
    155 352 CGPG8136 77933 SENSE
    156 353 CGPG8152 77619 SENSE
    157 354 CGPG8166 77621 SENSE
    158 355 CGPG8377 77629 SENSE
    159 356 CGPG8976 77832 SENSE
    160 357 CGPG8987 76802 SENSE
    161 358 CGPG9013 76829 SENSE
    162 359 CGPG9080 76961 SENSE
    163 360 CGPG9081 76973 SENSE
    164 361 CGPG9130 77150 SENSE
    165 362 CGPG9133 77186 SENSE
    166 363 CGPG9134 77103 SENSE
    167 364 CGPG9137 77139 SENSE
    168 365 CGPG9141 77187 SENSE
    169 366 CGPG9145 77140 SENSE
    170 367 CGPG9147 77164 SENSE
    171 368 CGPG9148 77176 SENSE
    172 369 CGPG9155 77165 SENSE
    173 370 CGPG9163 77166 SENSE
    174 371 CGPG9170 77155 SENSE
    175 372 CGPG9180 77180 SENSE
    176 373 CGPG9183 77121 SENSE
    177 374 CGPG9186 77157 SENSE
    178 375 CGPG9205 77195 SENSE
    179 376 CGPG9207 77124 SENSE
    180 377 CGPG9219 77261 SENSE
    181 378 CGPG9220 77273 SENSE
    182 379 CGPG9230 77203 SENSE
    183 380 CGPG9236 77275 SENSE
    184 381 CGPG9238 77204 SENSE
    185 382 CGPG9259 77266 SENSE
    186 383 CGPG9271 77220 SENSE
    187 384 CGPG9275 77268 SENSE
    188 385 CGPG9278 77209 SENSE
    189 386 CGPG9283 77269 SENSE
    190 387 CGPG9309 77451 SENSE
    191 388 CGPG9311 77452 SENSE
    192 389 CGPG9322 77430 SENSE
    193 390 CGPG9335 77432 SENSE
    194 391 CGPG9341 77433 SENSE
    195 392 CGPG9344 77444 SENSE
    196 393 CGPG9345 77409 SENSE
    197 394 CGPG976 12313 ANTI-
    SENSE
    10 207 CGPG1828 16322 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:197, 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:197 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 may be naturally occurring, including DNA from homologous genes from the same or a different species, or may 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 may 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 about 60% nucleotide equivalence; more preferably 70%; more preferably 80% equivalence; more preferably 85% equivalence; more preferably 90%; more preferably 95%; and/or more preferably 98% or 99% equivalence over a comparison window. A comparison window is preferably at least 50-100 nucleotides, and more preferably is the entire length of the polynucleotide provided herein. Optimal alignment of sequences for aligning a comparison window may be conducted by algorithms; preferably 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 may be a full-length molecule or a portion of a longer molecule. Preferentially, the window of comparison for determining polynucleotide identity of protein encoding sequences is the entire coding region.
  • Proteins useful for imparting improved traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins useful for generation of transgenic plants having improved traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 198 through SEQ ID NO: 394, as well as homologs of such proteins.
  • Homologs of the proteins useful in the invention are identified by comparison of the amino acid sequence of the protein to amino acid sequences of proteins from the same or different plant sources, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. As used herein, a homolog is a protein from the same or a different organism that performs the same biological function as the polypeptide to which it is compared. An orthologous relation between two organisms is not necessarily manifest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism phylogenetic separation, such as speciation. For a given protein, there may be no ortholog or more than one ortholog. Other complicating factors include alternatively spliced transcripts from the same gene, limited gene identification, redundant copies of the same gene with different sequence lengths or corrected sequence. 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. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal BLAST search is used in the present invention to filter hit sequences with significant E-values for ortholog identification. The reciprocal BLAST 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 is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. Thus, homolog is used herein to describe proteins that are assumed to have functional similarity by inference from sequence base similarity. The relationship of homologs with amino acid sequences of SEQ ID NO: 395 to SEQ ID NO: 19,938 to the proteins with amino acid sequences of SEQ ID NO: to 198 to SEQ ID NO: 394 are found in the listing of Table 2.
  • 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 comprises 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.
  • Homologs of the trait-improving proteins provided herein will generally demonstrate significant sequence identity. Of particular interest are proteins having at least 50% sequence identity, more preferably 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:198 through SEQ ID NO: 394. Of course useful proteins also include those with higher identity, e.g., 90% to 99% identity. Identity of protein homologs is determined by optimally 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: 198 through SEQ ID NO: 394.
  • 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 may 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 it is preferred that the promoter element in the DNA construct 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 HVA22 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, preferential 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 may also prevent gene function. For example, in many dicot plants, transformation with the T-DNA of Agrobacterium may 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 may 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.
  • In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the proteins disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include using glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
  • As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
  • mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified antifungal protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×107 to 2×108 lymphocytes.
  • The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp. 75-83). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
  • One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag-4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Spend virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).
  • Fusion procedures usually produce viable hybrids at low frequencies, about 1×10−6 to 1×10−8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azasenne blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
  • The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
    • Transformation Methods
  • Numerous methods for producing plant cell nuclei with recombinant DNA are known in the art and may 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); 5,591,616 (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.
  • In general it is preferred to introduce heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line. In special cases it may 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 implants include 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 are preferably 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, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may 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.
  • In practice DNA is introduced into only a small percentage of target cell nuclei in any one experiment. 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 transgenic DNA construct into their genomes. Preferred 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 may 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) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable 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 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.
  • Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, may 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 CO2, and 25-250 microeinsteins m−2 s−1 of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk 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 may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
  • Progeny may 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 improved traits to plants, Arabidopsis thaliana was transformed with a candidate recombinant DNA construct and screened for an improved trait.
  • Arabidopsis thaliana is used a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well-documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz, e.g., Methods in Arabidopsis Research e.g., (1992), World Scientific, New Jersey, New Jersey, in “Preface”).
  • A two-step screening process was employed which comprised 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 improved traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an improved trait. The following Table 3 summarizes the improved 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 improvement in a transgenic plant provided by the recombinant DNA. The codes for improved traits are:
  • “CK” which indicates cold tolerance improvement identified under a cold shock tolerance screen;
  • “CS” which indicates cold tolerance improvement identified by a cold germination tolerance screen;
  • “DS” which indicates drought tolerance improvement identified by a soil drought stress tolerance screen;
  • “PEG” which indicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen;
  • “HS” which indicates heat stress tolerance improvement identified by a heat stress tolerance screen;
  • “SS” which indicates high salinity stress tolerance improvement identified by a salt stress tolerance screen;
  • “LN” which indicates nitrogen use efficiency improvement 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 improved growth and development at early stages identified by an early plant growth and development screen;
  • “SP” which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein.
  • TABLE 3
    PEP Annotation
    SEQ Construct e %
    ID ID value id description trait
    198 11029 1.00E−170 85 gb|AAD02882.1|gamma-tocopherol DS LN
    methyltransferase [Arabidopsis
    thaliana]
    199 12223 / / / CK PEG
    200 10422 1.00E−133 100 gb|AAB47766.1|SNF5 homolog BSH LN
    [Arabidopsis thaliana]
    201 13485 4.00E−39 55 emb|CAB79347.1|hypothetical LN
    protein [Arabidopsis thaliana]
    202 13235 7.00E−76 100 emb|CAB78153.1|putative protein LN
    [Arabidopsis thaliana]
    203 13411 3.00E−29 100 ref|NP_179638.1|unknown protein LN CK
    [Arabidopsis thaliana]
    204 73944 0 95 ref|NP_189578.1|phosphorylase/ CS
    transferase, transferring glycosyl
    groups [Arabidopsis thaliana]
    205 13846 0 91 gb|AAN60291.1|unknown LN
    [Arabidopsis thaliana]
    206 12602 0 100 ref|NP_565974.1|PP2A-4; hydrolase/ LN
    protein phosphatase type 2A/protein
    serine/threonine phosphatase
    [Arabidopsis thaliana]
    207 74065 0 94 ref|NP_181390.1|DNA binding/ CS DS
    transcription factor [Arabidopsis
    thaliana] gb|AAN13033.1|putative
    elongation
    208 72783 0 83 ref|NP_564690.1|unknown protein CK PEG HS
    [Arabidopsis thaliana]
    209 17210 1.00E−92 100 ref|NP_563622.1|unknown protein CS CK PEG
    [Arabidopsis thaliana]
    210 72724 1.00E−128 90 ref|NP_190525.1|protein translocase/ LL
    protein transporter [Arabidopsis
    thaliana
    211 17805 1.00E−172 80 ref|NP_563761.1|unknown protein LN
    [Arabidopsis thaliana]
    212 16610 0 100 gb|AAL44550.1|fructose bisphosphate LN HS
    aldolase [Agrobacterium tumefaciens
    str. C58]
    213 76602 0 78 ref|NP_566515.1|signal transducer CS HS
    [Arabidopsis thaliana]
    214 18456 1.00E−135 81 ref|NP_176820.1|DNA binding/ CK SS PEG
    transcription factor [Arabidopsis
    thaliana]
    215 18414 1.00E−169 70 ref|NP_195583.1|translation initiation CK
    factor [Arabidopsis thaliana]
    216 71538 1.00E−137 90 emb|CAE85115.1|synaptotagmin SP
    [Arabidopsis thaliana]
    217 76532 0 99 ref|NP_564775.1|ATP binding/ CK CS
    carbohydrate binding/kinase/protein
    kinase/protein serine/threonine
    kinase/protein-tyrosine kinase/sugar
    binding [Arabidopsis thaliana]
    218 18231 1.00E−142 100 emb|CAC83762.1|allene oxide CK LN
    cyclase [Arabidopsis thaliana]
    219 18232 6.00E−85 89 ref|NP_191628.1|unknown protein CS
    [Arabidopsis thaliana]
    220 18256 1.00E−102 100 dbj|BAD44508.1|putative zinc finger SS CK HS
    protein (PMZ) [Arabidopsis thaliana]
    221 18258 1.00E−175 83 emb|CAA11525.1|transcription factor SP
    IIA large subunit [Arabidopsis
    thaliana]
    222 19193 1.00E−103 100 ref|NP_564682.1|lipid binding SP HS
    [Arabidopsis thaliana]
    dbj|BAE73268.1|xylogen like protein
    12 [Arabidopsis thaliana]
    223 71301 0 84 ref|NP_568393.1|nucleic acid binding LN
    [Arabidopsis thaliana]
    224 18354 0 96 ref|NP_567568.1|ATP binding/ LN
    kinase/protein kinase/protein
    serine/threonine kinase/protein-
    tyrosine kinase [Arabidopsis thaliana]
    225 77334 1.00E−117 100 ref|NP_178399.1|RNS1 LL
    (RIBONUCLEASE 1);
    endoribonuclease [Arabidopsis
    thaliana]
    226 19767 1.00E−180 95 gb|AAO69665.1|serine threonine CS DS
    protein phosphatase [Phaseolus
    acutifolius]
    227 19774 1.00E−100 57 ref|NP_974271.1|unknown protein CS PP
    [Arabidopsis thaliana]
    228 70992 1.00E−108 65 gb|ABE87200.1|hypothetical protein CS PEG
    MtrDRAFT_AC151668g4v1
    [Medicago truncatula]
    229 19956 1.00E−146 100 gb|AAF64040.1|14-3-3-like protein PP
    [Glycine max]
    230 70948 3.00E−59 45 ref|NP_201297.1|CIP8 (COP1- PP HS
    INTERACTING PROTEIN 8);
    protein binding/zinc ion binding
    [Arabidopsis thaliana]
    231 70201 0 68 ref|NP_567072.1|ATP binding/ CS SS
    kinase/protein kinase/protein
    serine/threonine kinase/protein-
    tyrosine kinase [Arabidopsis thaliana]
    232 19973 0 96 gb|AAB04057.1|S-adenosyl-L- PP SS
    methionine:delta24-sterol-C-
    methyltransferase
    233 70987 0 93 gb|AAM83095.1|SOS2-like protein CK CS HS SS PEG
    kinase [Glycine max]
    234 70950 1.00E−116 77 dbj|BAD38167.1|putative leucine PP PEG
    zipper protein [Oryza sativa (japonica
    cultivar-group)]
    235 70962 1.00E−179 78 gb|ABE93200.1|conserved PP HS
    hypothetical protein [Medicago
    truncatula]
    236 70915 0 76 dbj|BAE71210.1|hypothetical protein CS PP
    [Trifolium pratense] dbj|BAE71208.1|
    hypothetical protein [Trifolium
    pratense]
    237 19947 1.00E−116 65 gb|AAS38575.1|short-chain CS DS SS
    dehydrogenase Tic32 [Pisum
    sativum]
    238 70969 1.00E−144 76 ref|NP_174321.2|unknown protein SS
    [Arabidopsis thaliana]
    239 70985 0 69 dbj|BAB08296.1|unnamed protein CS HS PEG
    product [Arabidopsis thaliana]
    sp|Q9FMT4|Y5417_ARATH Protein
    At5g14170
    240 70971 1.00E−92 63 gb|ABE80664.1|GNS1/SUR4 SS
    membrane protein [Medicago
    truncatula] gb|ABE78502.1|
    GNS1/SUR4 membrane protein
    [Medicago truncatula]
    241 70963 1.00E−96 56 ref|NP_177178.1|CYCD1; 1; cyclin- CS PP HS
    dependent protein kinase regulator
    [Arabidopsis thaliana]
    242 70994 2.00E−33 44 ref|NP_172962.1|RHA2A; protein PP DS HS SS
    binding/ubiquitin-protein ligase/zinc
    ion binding [Arabidopsis thaliana]
    243 70956 0 85 gb|AAO23063.1|ent-kaurenoic acid CS SS HS
    oxidase [Pisum sativum]
    244 70995 1.00E−132 66 dbj|BAD73789.1|putative CK CS PP LL PEG
    uncharacterized hypothalamus protein
    HT010 [Oryza sativa (japonica
    cultivar-group)]
    245 73306 0 93 ref|NP_201243.1|phosphoric ester SS PEG
    hydrolase [Arabidopsis thaliana]
    246 70657 9.00E−31 73 emb|CAB78150.1|probable wound- SP LL
    induced protein [Arabidopsis
    thaliana]
    247 70660 2.00E−32 79 ref|NP_568217.1|transcription SP
    regulator [Arabidopsis thaliana]
    248 74536 0 91 ref|NP_001031061.1|PGDH; CS
    phosphoglycerate dehydrogenase
    [Arabidopsis thaliana]]
    249 71328 0 99 gb|AAD46022.1|Strong simlarity to PP
    gb|286426 F10M6.190 cytochrome
    p450 homolog from Arabidopsis
    thaliana BAC
    250 71329 0 94 ref|NP_177477.1|heme binding/iron LN
    ion binding/monooxygenase/oxygen
    binding [Arabidopsis thaliana]
    251 70755 1.00E−142 100 ref|NP_188925.1|unknown protein LL
    [Arabidopsis thaliana]
    252 70684 3.00E−53 100 emb|CAB80194.1|putative protein CS LN HS PP
    [Arabidopsis thaliana]
    253 73330 1.00E−158 72 emb|CAB96855.1|putative protein CK CS SS
    [Arabidopsis thaliana]
    254 76202 3.00E−83 67 emb|CAA16586.1|putative protein LL
    [Arabidopsis thaliana]
    255 72807 0 100 ref|NP_173478.2|nucleotide binding PEG CK HS SS
    [Arabidopsis thaliana]
    256 72811 1.00E−176 100 ref|NP_565782.1|nucleotide binding CK SS PEG
    [Arabidopsis thaliana]
    257 73235 0 89 ref|NP_564867.1|pepsin A SS HS
    [Arabidopsis thaliana]
    258 72813 2.00E−79 93 ref|NP_850506.1|unknown protein LL LN
    [Arabidopsis thaliana]
    259 72825 1.00E−154 95 ref|NP_563635.1|oxidoreductase CK PEG HS
    [Arabidopsis thaliana]
    260 73628 1.00E−163 92 ref|NP_849428.1|oxidoreductase CS DS LL HS PEG
    [Arabidopsis thaliana]
    261 76105 0 93 emb|CAB79123.1|receptor kinase-like DS
    protein [Arabidopsis thaliana]
    262 73242 0 100 ref|NP_567292.1|unknown protein SP PEG
    [Arabidopsis thaliana]
    263 74217 0 100 emb|CAB93726.1|cytochrome P450- DS CK
    like protein [Arabidopsis thaliana]
    264 73735 0 97 gb|AAU94404.1|At3g48520 CK
    [Arabidopsis thaliana]
    265 73256 1.00E−114 77 ref|NP_566329.1|unknown protein CS PP PEG
    [Arabidopsis thaliana]
    gb|AAL06975.1|
    266 73260 4.00E−37 100 ref|NP_566488.1|unknown protein CS
    [Arabidopsis thaliana]
    267 75822 1.00E−90 88 emb|CAB63015.1|putative protein LL
    [Arabidopsis thaliana]
    268 72001 0 96 ref|NP_200045.1|CYP96A4; heme CS PP HS
    binding/iron ion binding/
    monooxygenase/oxygen binding
    [Arabidopsis thaliana]
    269 72056 0 92 ref|NP_010186.1|Essential, non- SP
    ATPase regulatory subunit of the 26S
    proteasome lid required for the
    assembly and activity of the 26S
    proteasome;
    270 77308 0 100 ref|NP_178642.1|SCPL38; serine PEG
    carboxypeptidase [Arabidopsis
    thaliana]
    271 73766 1.00E−163 100 emb|CAB62363.1|MTN3-like protein LL
    [Arabidopsis thaliana]
    272 72774 1.00E−139 95 ref|NP_011052.1|Constituent of 66S LL CK HS
    pre-ribosomal particles, involved in
    60S ribosomal subunit biogenesis;
    273 72788 0 81 ref|NP_010007.1|General repressor of CK CS SS
    transcription, forms complex with
    Cyc8p, involved in the establishment
    of repressive chromatin structure
    through interactions with histones H3
    and H4, appears to enhance
    expression of some genes
    274 72753 1.00E−105 85 ref|NP_013125.1|One of four subunits CS PP HS PEG
    of the endosomal sorting complex
    required for transport III (ESCRT-
    III);
    275 72709 0 94 ref|NP_011827.1|Low affinity LL
    methionine permease, similar to
    Mup1p;
    276 73954 0 98 ref|NP_200954.1|MIM; ATP binding HS
    [Arabidopsis thaliana]
    277 73137 1.00E−110 94 gb|AAD32800.1|putative thioredoxin CS
    H [Arabidopsis thaliana]
    278 73161 1.00E−36 65 emb|CAB85993.1|putative protein CS PEG
    [Arabidopsis thaliana]
    279 73057 1.00E−133 95 ref|NP_012770.1|Tetrameric SP
    phosphoglycerate mutase, mediates
    the conversion of 3-phosphoglycerate
    to 2-phosphoglycerate during
    glycolysis and the reverse reaction
    during gluconeogenesis;
    280 73127 0 99 dbj|BAB05414.1|aspartate SS HS
    aminotransferase [Bacillus halodurans
    C-125] ref|NP_242561.1|aspartate
    aminotransferase [Bacillus halodurans
    C-125]
    281 73033 0 96 dbj|BAB07276.1|2,3- CK
    bisphosphoglycerate-independent
    phosphoglycerate mutase [Bacillus
    halodurans C-125]
    282 73105 0 99 emb|CAB13630.1|glutamine PP PEG
    synthetase [Bacillus subtilis subsp.
    subtilis str. 168]
    283 73141 0 95 ref|NP_414696.1|glutamate-1- CS
    semialdehyde aminotransferase
    [Escherichia coli K12]
    284 73165 0 99 ref|NP_288110.1|pyruvate kinase DS PEG
    [Escherichia coli O157:H7 EDL933]
    285 73155 1.00E−173 92 emb|CAC47346.1|PROBABLE CS
    FRUCTOSE-BISPHOSPHATE
    ALDOLASE CLASS I PROTEIN
    [Sinorhizobium meliloti]]
    286 73026 1.00E−116 87 emb|CAG77169.1|triosephosphate PEG
    isomerase [Erwinia carotovora subsp.
    atroseptica SCRI1043]
    287 73120 8.00E−82 100 ref|NP_441918.1|nucleoside CS SS HS
    diphosphate kinase [Synechocystis sp.
    PCC 6803]
    288 73133 2.00E−63 90 gb|ABA76342.1|Nucleoside LL
    diphosphate kinase [Pseudomonas
    fluorescens PfO-1]
    289 73134 9.00E−98 81 emb|CAE15979.1|ribose 5-phosphate SS CK
    isomerase A (phosphoriboisomerase
    A) [Photorhabdus luminescens subsp.
    laumondii TTO1]
    290 73123 0 95 ref|NP_180412.2|nucleic acid binding CS PEG
    [Arabidopsis thaliana]
    291 73981 0 93 ref|NP_014797.1|hypothetical protein; DS PEG
    Slp1p [Saccharomyces cerevisiae]
    292 73136 0 91 ref|NP_012454.1|Nuclear actin- DS PP
    related protein involved in chromatin
    remodeling
    293 73172 0 100 ref|NP_04952.1|Protein involved in CK PEG HS
    ER-to-Golgi transport; Sly41p
    [Saccharomyces cerevisiae]
    294 73020 0 93 ref|NP_014993.1|Proline permease, LL
    required for high-affinity transport of
    proline
    295 72946 0 95 ref|NP_012534.1|Vacuolar PEG LL
    transporter, imports large neutral
    amino acids into the vacuole
    296 74746 0 93 ref|NP_566298.1|ATP binding/ CK
    kinase/protein kinase/protein
    serine/threonine kinase/protein-
    tyrosine kinase [Arabidopsis thaliana]
    297 77312 0 90 ref|NP_178463.1|CDC48B; ATP PP SS HS
    binding/ATP-dependent peptidase/
    ATPase/nucleoside-triphosphatase/
    nucleotide binding/serine-type
    endopeptidase [Arabidopsis thaliana]
    298 75237 0 100 ref|NP_173390.1|kinase [Arabidopsis LN
    thaliana] gb|AAF98405.1|Unknown
    protein [Arabidopsis thaliana]
    299 75240 0 100 emb|CAB69854.1|putative protein CK
    [Arabidopsis thaliana]
    300 74349 1.00E−148 79 ref|NP_565187.1|transcription LN
    regulator [Arabidopsis thaliana]
    301 76422 5.00E−44 100 ref|NP_171781.1|unknown protein LN
    [Arabidopsis thaliana]
    302 70812 1.00E−152 92 ref|NP_850182.1|PUR ALPHA-1; PP PEG
    nucleic acid binding [Arabidopsis
    thaliana]
    303 77322 1.00E−172 100 gb|AAW38983.1|At5g10750 LN LL
    [Arabidopsis thaliana]
    304 74662 4.00E−89 100 ref|NP_001031939.1|ubiquitin PP HS
    conjugating enzyme/ubiquitin-like
    activating enzyme [Arabidopsis
    thaliana]
    305 76527 0 96 emb|CAB51062.1|cell division cycle CS
    protein 23 homolog [Arabidopsis
    thaliana]
    306 77020 0 98 emb|CAB75781.1|putative transporter CS
    protein [Arabidopsis thaliana]
    ref|NP_190154.1|transporter
    [Arabidopsis thaliana]
    307 77609 0 96 gb|AAU95452.1|At5g04420 LL SS
    [Arabidopsis thaliana]
    308 73485 0 100 emb|CAD84238.1|Glyceraldehyde 3- PEG
    phosphate dehydrogenase
    309 73433 1.00E−132 65 ref|ZP_00819153.1|putative alcohol SP LN PP
    dehydrogenase [Marinobacter
    aquaeolei VT8]
    310 73411 0 99 emb|CAB14878.1|pyruvate kinase CS HS PP PEG
    [Bacillus subtilis subsp. subtilis str.
    168]
    311 73568 0 97 emb|CAC41401.1|PROBABLE SP
    SUCCINATE-SEMIALDEHYDE
    DEHYDROGENASE [NADP+]
    PROTEIN [Sinorhizobium meliloti]
    312 74688 0 98 emb|CAA16688.1|receptor protein CS PP SS PEG
    kinase - like protein [Arabidopsis
    thaliana]
    313 74420 1.00E−142 99 emb|CAD85691.1|Phosphoglycerate PP LN SS
    mutase family [Nitrosomonas
    europaea ATCC 19718]
    314 74435 1.00E−117 100 emb|CAC41549.1|PROBABLE LL
    PHOSPHOGLYCERATE MUTASE
    1 PROTEIN [Sinorhizobium meliloti]
    315 74460 0 95 ref|NP_441738.1|fructose-1,6- CK
    bisphosphatase [Synechocystis sp.
    PCC 6803]
    316 74566 0 93 emb|CAC48499.1|Putative trehalose SP PP SS PEG HS
    synthase protein [Sinorhizobium
    meliloti 1021]
    317 77610 0 94 gb|AAC23406.1|hypothetical protein CS HS
    [Arabidopsis thaliana]
    318 77618 0 100 ref|NP_850453.1|JAR1 PP HS
    (JASMONATE RESISTANT 1)
    [Arabidopsis thaliana]
    319 77517 7.00E−77 100 emb|CAB75802.1|putative protein CS LL
    [Arabidopsis thaliana]
    320 77518 1.00E−49 100 ref|NP_199600.1|oxidoreductase, SS LL HS
    acting on NADH or NADPH, quinone
    or similar compound as acceptor
    [Arabidopsis thaliana]
    321 76460 / / / CS PEG
    322 77069 0 85 ref|NP_201196.1|unknown protein PP
    [Arabidopsis thaliana]
    323 76161 1.00E−161 100 ref|NP_851282.1|unknown protein CK
    [Arabidopsis thaliana]
    324 76171 1.00E−178 95 ref|NP_176204.1|oxidoreductase LL
    [Arabidopsis thaliana]
    325 76178 5.00E−60 86 ref|NP_565029.1|unknown protein CS PP HS LL PEG
    [Arabidopsis thaliana]
    326 76467 6.00E−86 89 ref|NP_565671.1|unknown protein LL
    [Arabidopsis thaliana]
    327 77536 1.00E−119 83 gb|AAS99692.1|At1g10020 SP CS DS PP LN
    [Arabidopsis thaliana]
    328 76576 1.00E−145 100 ref|NP_181023.1|FAH1 (FATTY DS
    ACID HYDROXYLASE 1); catalytic
    [Arabidopsis thaliana]
    329 74862 1.00E−145 100 gb|ABA77057.1|Delta 1-pyrroline-5- PP
    carboxylate reductase [Pseudomonas
    fluorescens PfO-1]
    330 74863 1.00E−93 100 ref|NP_012420.1|Nucleosome LL PEG CK SS
    assembly factor, involved in
    chromatin assembly after DNA
    replication]
    331 74858 5.00E−50 68 gb|AAK14395.1|response regulator LL
    protein [Dianthus caryophyllus]
    332 74933 1.00E−177 95 gb|AAK85899.1|AGR_C_118p LL
    [Agrobacterium tumefaciens str. C58]
    333 75379 1.00E−105 76 dbj|BAD73205.1|unknown protein LN
    [Oryza sativa (japonica cultivar-
    group)]
    334 77816 4.00E−41 89 gb|ABA98984.1|expressed protein SP PP HS
    [Oryza sativa (japonica cultivar-
    group)]
    335 75434 3.00E−52 79 ref|XP_472650.1|OSJNBa0027P08.15 LN
    [Oryza sativa (japonica cultivar-
    group)]
    336 77821 0 71 gb|ABE84883.1|conserved PP SS HS PEG
    hypothetical protein [Medicago
    truncatula]
    337 75685 0 82 ref|XP_475937.1|unknown protein LL
    [Oryza sativa (japonica cultivar-
    group)]
    338 75654 0 100 ref|NP_563865.1|unknown protein SP CK HS
    [Arabidopsis thaliana]
    339 75692 1.00E−119 87 dbj|BAD30296.1|peptidyl-prolyl cis- CK LN
    trans isomerase-like protein [Oryza
    sativa (japonica cultivar-group)]
    340 75657 6.00E−57 67 dbj|BAD38392.1|DNAJ heat shock N- CS LL HS
    terminal domain-containing protein-
    like [Oryza sativa (japonica cultivar-
    group)]
    341 75622 1.00E−30 78 dbj|BAD45825.1|unknown protein LN
    [Oryza sativa (japonica cultivar-
    group)]
    342 77549 1.00E−132 89 emb|CAB80891.1|AT4g00820 PP HS
    [Arabidopsis thaliana]]
    343 77917 0 91 ref|NP_197917.1|EBF2 (EIN3- PP PEG
    BINDING F BOX PROTEIN 2)
    [Arabidopsis thaliana]
    344 77568 9.00E−99 76 ref|NP_849792.1|nucleic acid binding PP
    [Arabidopsis thaliana]
    345 77570 7.00E−54 100 gb|AAS75309.1|multidomain CK SS
    cyclophilin type peptidyl-prolyl cis-
    trans isomerase [Arabidopsis
    thaliana]]
    346 77338 4.00E−24 65 gb|AAM61454.1|unknown PEG
    [Arabidopsis thaliana]
    347 77580 3.00E−45 100 dbj|BAB01457.1|unnamed protein PEG CS HS SS
    product [Arabidopsis thaliana]
    348 77928 4.00E−38 100 ref|NP_196244.1|unknown protein HS
    [Arabidopsis thaliana]
    349 77349 1.00E−81 93 emb|CAC05463.1|putative lipid LL SS
    transfer protein [Arabidopsis thaliana]
    350 77357 1.00E−155 95 ref|NP_175357.1|unknown protein PEG
    [Arabidopsis thaliana]
    351 77587 / / / CS PP HS SS
    352 77933 / / / SS
    353 77619 3.00E−40 98 dbj|BAB09403.1|unnamed protein CK SS PEG HS
    product [Arabidopsis thaliana]
    354 77621 4.00E−58 74 ref|NP_197632.1|Rac GTPase CK
    activator [Arabidopsis thaliana]
    [Arabidopsis thaliana]
    355 77629 5.00E−36 100 ref|NP_196372.1|GRP19 [Arabidopsis SS HS
    thaliana]
    356 77832 0 83 ref|NP_917762.1|P0501G01.24 CS SS PEG HS LN
    [Oryza sativa (japonica cultivar-
    group)]
    357 76802 0 76 ref|NP_181908.1|actin binding CK
    [Arabidopsis thaliana]
    gb|AAB64026.1|unknown protein
    [Arabidopsis thaliana]
    358 76829 0 89 ref|NP_909912.1|ferredoxin-NADP+ CK
    reductase [Oryza sativa]
    359 76961 0 65 ref|XP_473189.1|OSJNBa0073E02.11 LL LN
    [Oryza sativa (japonica cultivar-
    group)]
    360 76973 1.00E−151 76 ref|XP_480055.1|unknown protein LL
    [Oryza sativa (japonica cultivar-
    group)]
    361 77150 0 76 dbj|BAD61385.1|putative SP HS PEG
    nucleostemin [Oryza sativa (japonica
    cultivar-group)]
    362 77186 0 85 dbj|BAD27898.1|putative PP
    pentatricopeptide (PPR) repeat-
    containing protein [Oryza sativa
    (japonica cultivar-group)]
    363 77103 1.00E−174 66 ref|NP_568580.1|catalytic LL DS
    [Arabidopsis thaliana]
    364 77139 2.00E−93 58 ref|NP_171690.1|PFC1 (PALEFACE CS SS
    1) [Arabidopsis thaliana]
    365 77187 1.00E−160 83 ref|NP_913437.1|3-methyl-2- CS PP LL PEG
    oxobutanoate hydroxy-methyl-
    transferase-like protein [Oryza sativa
    (japonica cultivar-group)]
    366 77140 0 77 dbj|BAD36145.1|membrane protein CK PEG SS
    PTM1-like [Oryza sativa (japonica
    cultivar-group)]
    367 77164 0 91 gb|ABF93778.1|DNA polymerase PP HS
    delta small subunit, putative,
    expressed [Oryza sativa (japonica
    cultivar-group)]
    368 77176 0 76 ref|NP_914476.1|putative LL
    phytochrome P450 [Oryza sativa
    (japonica cultivar-group)]
    369 77165 1.00E−139 75 ref|NP_914949.1|serine/threonine CK LL LN
    protein kinase-like protein [Oryza
    sativa (japonica cultivar-group)]
    370 77166 0 85 dbj|BAB75233.1|all3534 [Nostoc sp. CK PP
    PCC 7120]
    371 77155 0 97 emb|CAA35550.1|hycE [Escherichia PP
    coli]
    372 77180 2.00E−62 61 gb|ABA99663.1|expressed protein CS PP HS PEG
    [Oryza sativa (japonica cultivar-
    group)]
    373 77121 1.00E−166 93 dbj|BAB05404.1|transcriptional CK
    repressor of the biotin operon
    [Bacillus halodurans C-125]
    374 77157 0 100 ref|NP_531453.1|3,4-dihydroxy-2- CK LL
    butanone-4-phoshate synthase/GTP
    cyclohydrolase II [Agrobacterium
    tumefaciens str. C58]
    375 77195 0 94 gb|AAM71555.1|mannose-6- PP SS PEG LL
    phosphate isomerase/mannose-1-
    phosphate guanylyl transferase
    [Chlorobium tepidum TLS]
    376 77124 1.00E−180 100 ref|NP_418404.1|biotin--protein PP SS PEG
    ligase [Escherichia coli K12]
    377 77261 0 100 ref|NP_416982.1|hydrogenase 4, PEG
    subunit [Escherichia coli K12]
    378 77273 0 93 dbj|BAB06534.1|glycine PP PEG
    dehydrogenase subunit 1 [Bacillus
    halodurans C-125]
    379 77203 1.00E−176 60 dbj|BAD33942.1|putative serine PEG CK
    carboxypeptidase precursor [Oryza
    sativa (japonica cultivar-group)]
    380 77275 4.00E−37 55 gb|ABA91490.1|expressed protein SS HS PEG
    [Oryza sativa (japonica cultivar-
    group)]
    381 77204 2.00E−27 44 ref|XP_469963.1|putative protease CK CS
    inhibitor [Oryza sativa (japonica
    cultivar-group)]
    382 77266 7.00E−25 73 gb|AAT93978.1|unknown protein CS PP PEG
    [Oryza sativa (japonica cultivar-
    group)]
    383 77220 1.00E−169 78 dbj|BAD52854.1|putative non- LL
    phototropic hypocotyl 3 [Oryza sativa
    (japonica cultivar-group)]
    384 77268 4.00E−71 85 gb|ABF95596.1|ETC complex I PP SS
    subunit conserved region family
    protein, expressed [Oryza sativa
    (japonica cultivar-group)]
    385 77209 9.00E−86 85 ref|XP_479456.1|putative 60S PP LL
    ribosome subunit biogenesis protein
    [Oryza sativa (japonica cultivar-
    group)]
    386 77269 2.00E−56 70 dbj|BAD32031.1|unknown protein SS
    [Oryza sativa (japonica cultivar-
    group)]
    387 77451 0 71 ref|XP_475231.1|putative PP SS
    microtubule-associated protein [Oryza
    sativa (japonica cultivar-group)]
    388 77452 1.00E−106 69 dbj|BAD69045.1|unknown protein PP
    [Oryza sativa (japonica cultivar-
    group)]
    389 77430 1.00E−160 83 dbj|BAD33328.1|putative protein CS PP SS
    serine/threonine kinase [Oryza sativa
    (japonica cultivar-group)]
    390 77432 0 86 ref|NP_849565.1|carbohydrate PP HS PEG
    transporter/nucleoside transporter/
    sugar porter [Arabidopsis thaliana]
    gb|AAM19835.1|
    AT4g35300/F23E12_140
    [Arabidopsis thaliana]
    391 77433 0 100 gb|AAK59487.1|putative cleavage CK PP
    and polyadenylation specificity factor
    [Arabidopsis thaliana]
    392 77444 0 96 ref|NP_199947.1|unknown protein CS PP
    [Arabidopsis thaliana]
    393 77409 0 91 ref|NP_001032163.1|unknown protein PP
    [Arabidopsis thaliana]
    394 12313 0 94 ref|NP_189150.1|QUA1 SP
    (QUASIMODO1); transferase,
    transferring glycosyl groups/
    transferase, transferring hexosyl
    groups [Arabidopsis thaliana]
    • Trait Improvement Screens
  • DS-Improvement 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 may find particular use 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-Improvement 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-Improvement 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 improved 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-Improvement 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 may also impart improved 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-Improvement of tolerance to cold stress: Low temperature may 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.
  • Improvement 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: 231 can be used to improve both salt stress tolerance and cold stress tolerance in plants. Of particular interest, plants transformed with PEP SEQ ID NO: 233 can resist heat stress, salt stress and cold stress. In addition to these multiple stress tolerance genes, the stress tolerance conferring genes provided by the present invention may be used in combinations to generate transgenic plants that can resist multiple stress conditions.
  • PP-Improvement 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 may also be used to generate transgenic plants that are more resistant to various stress conditions due to improved 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: 268 which can improve the plant early growth and development, and impart heat and cold 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-Improvement 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 improved development during leaf development and seed maturation providing a higher yield potential as compared to control plants.
  • LL-Improvement 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 may be suitable for a higher density planting, thereby resulting increased yield per unit area.
  • LN-Improvement 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.
  • According to the present invention, 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 may recognize that the transgenic plants provided by the present invention with enhanced nitrogen use efficiency may also have altered amino acid or protein compositions, increased yield and/or better seed quality. The transgenic plants of the present invention may 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 may 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 improved 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 improved 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: 197, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO: 198 through SEQ ID NO: 394. In another aspect, the present invention provides the protein sequences of identified homologs for a sequence listed as SEQ ID NO: 395 through SEQ ID NO: 19938. 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 improved traits provided by the present invention are not limited to any particular plant species. Indeed, the plants according to the present invention may be of any plant species, i.e., may be monocotyledonous or dicotyledonous. Preferably, 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 improved 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 improved traits as the transgenic Arabidopsis counterpart. Improved physiological properties in transgenic plants of the present invention may be confirmed in responses to stress conditions, for example in assays using imposed stress conditions to detect improved responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions. Biomass measures may be made on greenhouse or field grown plants and may 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 may 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 may 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 may 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 may 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 Improved 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/m2/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
    Drought Seed Time to wilting
    PEP score yield Risk
    SEQ Construct Nomination Delta P- Delta P- score P-
    ID NO ID ID Orientation mean value mean value mean value
    198 11029 CGPG106 SENSE 0.109 0.381 0.072 0.717 0.119 1.000
    292 73136 CGPG5764 SENSE 0.030 0.090 0.136 0.067 0.104 1.000
    284 73165 CGPG5661 SENSE −0.031 0.504 0.526 0.018 −0.075 1.000
    260 73628 CGPG5025 SENSE 0.342 0.038 −0.474 0.022 0.208 1.000
    291 73981 CGPG5757 SENSE 0.517 0.026 −0.096 0.000 0.573 1.000
    207 74065 CGPG1828 ANTI- −0.078 0.164 0.646 0.027 0.048 1.000
    SENSE
    263 74217 CGPG5144 SENSE −0.026 0.414 0.418 0.032 0.071 1.000
    261 76105 CGPG5041 SENSE 0.288 0.029 −1.780 0.027 0.173 1.000
    328 76576 CGPG7281 SENSE 0.241 0.032 −0.555 0.442 0.212 1.000
    327 77536 CGPG7272 SENSE 0.089 0.194 1.059 0.001 0.042 1.000
    226 19767 CGPG3918 SENSE 0.117 0.038 0.164 0.296 / /
    237 19947 CGPG4069 SENSE −0.009 0.852 −0.104 0.504 / /
    242 70994 CGPG4122 SENSE 0.040 0.021 −0.131 0.447 / /
    363 77103 CGPG9134 SENSE 0.145 0.023 −0.499 0.096 / /
  • If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance)
  • If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 226, 237, 242, or 363 showed improved drought tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.
    • 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/m2/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
    Root length Growth stage weight at
    at day 14 at day 14 day 14
    PEP Construct Nomination Delta Risk score Delta
    SEQ ID ID ID Orientation mean P-value mean P-value mean P-value
    239 70985 CGPG4088 SENSE 0.480 0.036 1.176 0.196 1.333 0.018
    233 70987 CGPG4048 SENSE 0.228 0.021 −0.036 0.670 1.214 0.002
    268 72001 CGPG5221 SENSE 0.273 0.004 0.210 0.330 1.393 0.009
    274 72753 CGPG5540 SENSE 0.434 0.006 0.414 0.258 1.100 0.006
    276 73954 CGPG5577 SENSE 0.406 0.002 0.455 0.282 1.274 0.000
    325 76178 CGPG7225 SENSE 0.714 0.001 1.093 0.145 1.537 0.000
    361 77150 CGPG9130 SENSE 0.219 0.032 0.178 0.237 1.159 0.002
    367 77164 CGPG9147 SENSE 0.317 0.026 0.651 0.099 1.152 0.002
    390 77432 CGPG9335 SENSE 0.581 0.001 0.457 0.124 1.551 0.000
    351 77587 CGPG8107 SENSE 0.488 0.006 0.628 0.072 1.328 0.001
    348 77928 CGPG8082 SENSE 0.462 0.032 0.191 0.301 1.498 0.004
    212 16610 CGPG2499 SENSE 0.055 0.640 0.109 0.609 0.983 0.020
    220 18256 CGPG3363 SENSE 0.070 0.138 0.071 0.541 1.021 0.004
    222 19193 CGPG3375 SENSE 0.255 0.094 0.267 0.368 1.023 0.003
    252 70684 CGPG4588 SENSE 0.010 0.781 0.003 0.979 1.002 0.045
    230 70948 CGPG3990 SENSE 0.282 0.095 0.351 0.335 1.045 0.023
    243 70956 CGPG4140 SENSE 0.155 0.313 0.091 0.320 1.129 0.018
    235 70962 CGPG4057 SENSE 0.469 0.085 0.267 0.508 1.410 0.007
    241 70963 CGPG4121 SENSE −0.002 0.979 −0.131 0.311 0.976 0.005
    242 70994 CGPG4122 SENSE 0.140 0.574 1.204 0.096 1.207 0.030
    244 70995 CGPG4154 SENSE 0.374 0.112 1.640 0.171 1.328 0.013
    208 72783 CGPG2206 SENSE 0.387 0.057 0.053 0.509 1.013 0.019
    255 72807 CGPG4912 SENSE 0.357 0.071 0.443 0.020 1.099 0.009
    259 72825 CGPG5001 SENSE 0.171 0.349 0.213 0.234 1.118 0.012
    287 73120 CGPG5704 SENSE 0.096 0.490 0.193 0.364 1.080 0.033
    280 73127 CGPG5640 SENSE −0.220 0.077 −0.194 0.030 0.643 0.014
    293 73172 CGPG5783 SENSE 0.283 0.197 0.887 0.162 1.154 0.007
    310 73411 CGPG6440 SENSE 0.198 0.389 0.631 0.345 1.320 0.003
    260 73628 CGPG5025 SENSE 0.406 0.149 1.475 0.365 1.169 0.016
    316 74566 CGPG6796 SENSE 0.627 0.092 0.777 0.466 1.714 0.014
    338 75654 CGPG7804 SENSE 0.292 0.237 0.804 0.315 0.940 0.017
    340 75657 CGPG7828 SENSE 0.390 0.067 1.222 0.307 1.521 0.011
    213 76602 CGPG2653 SENSE 0.036 0.748 0.092 0.670 0.752 0.025
    372 77180 CGPG9180 SENSE 0.068 0.555 −0.045 0.177 0.871 0.022
    380 77275 CGPG9236 SENSE 0.282 0.125 0.971 0.285 1.027 0.013
    297 77312 CGPG5927 SENSE 0.201 0.344 0.710 0.372 1.039 0.032
    320 77518 CGPG6953 SENSE 0.238 0.344 0.069 0.655 1.188 0.015
    342 77549 CGPG7933 SENSE 0.184 0.180 −0.012 / 0.978 0.007
    347 77580 CGPG8062 SENSE 0.157 0.143 −0.074 / 0.891 0.049
    317 77610 CGPG6805 SENSE 0.023 0.937 −0.063 / 1.207 0.029
    318 77618 CGPG6810 SENSE 0.002 0.942 −0.074 / 1.015 0.006
    353 77619 CGPG8152 SENSE 0.213 0.316 −0.076 0.457 0.956 0.019
    355 77629 CGPG8377 SENSE 0.095 0.124 −0.063 / 1.133 0.005
    334 77816 CGPG7529 SENSE 0.033 0.705 0.856 0.385 0.611 0.038
  • If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 307, 313, 327, 330, 349, 366, or 387 showed improved heat stress tolerance evidenced by the second criteria 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 experiment 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/m2. 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
    Growth Seedling
    Root length Root length stage at weight at
    at day 11 at day 14 day 14 day 14
    PEP Construct Delta Delta Delta Delta
    SEQ ID ID Orientation mean P-value mean P-value mean P-value mean P-value
    220 18256 SENSE 0.192 0.232 0.236 0.080 1.291 0.151 0.598 0.039
    214 18456 SENSE 0.213 0.089 0.209 0.197 2.062 0.197 0.476 0.046
    232 19973 SENSE 0.289 0.012 0.299 0.002 0.976 0.054 1.002 0.013
    231 70201 SENSE 0.363 0.030 0.403 0.052 2.083 0.229 1.057 0.014
    243 70956 SENSE 0.006 0.923 0.128 0.016 −0.045 NA −0.364 0.371
    238 70969 SENSE 0.435 0.091 0.471 0.027 0.653 0.473 0.994 0.058
    240 70971 SENSE 0.337 0.063 0.257 0.041 1.418 0.226 0.593 0.030
    233 70987 SENSE 0.363 0.030 0.284 0.044 1.434 0.380 0.865 0.012
    273 72788 SENSE 0.484 0.019 0.456 0.016 1.813 0.314 0.929 0.008
    256 72811 SENSE 0.385 0.024 0.409 0.002 2.968 0.031 0.805 0.004
    287 73120 SENSE 0.341 0.001 0.244 0.019 0.613 0.103 0.755 0.015
    280 73127 SENSE 0.501 0.028 0.395 0.055 0.212 0.175 0.739 0.023
    289 73134 SENSE 0.226 0.134 0.266 0.045 0.152 0.487 0.378 0.056
    257 73235 SENSE 0.431 0.020 0.360 0.018 1.618 0.327 0.650 0.009
    245 73306 SENSE 0.228 0.046 0.219 0.018 0.249 0.217 0.708 0.008
    253 73330 SENSE 0.196 0.225 0.186 0.029 0.000 NA 0.543 0.045
    316 74566 SENSE 0.331 0.001 0.076 0.422 0.597 0.031 0.716 0.003
    312 74688 SENSE 0.480 0.076 0.415 0.031 0.000 NA 0.808 0.062
    376 77124 SENSE 0.340 0.034 0.322 0.015 2.306 0.137 0.411 0.168
    364 77139 SENSE 0.339 0.096 0.287 0.041 1.683 0.294 0.602 0.039
    375 77195 SENSE 0.445 0.009 0.345 0.010 0.367 0.185 0.582 0.022
    384 77268 SENSE 0.495 0.047 0.468 0.102 1.906 0.079 0.992 0.042
    386 77269 SENSE 0.454 0.038 0.426 0.004 1.996 0.065 1.136 0.001
    380 77275 SENSE 0.457 0.008 0.487 0.040 2.119 0.154 1.070 0.010
    297 77312 SENSE 0.335 0.086 0.361 0.013 0.960 0.208 0.723 0.063
    389 77430 SENSE 0.303 0.030 0.360 0.004 0.131 0.495 0.894 0.022
    320 77518 SENSE 0.171 0.017 0.185 0.026 −0.134 0.443 0.339 0.109
    345 77570 SENSE 0.326 0.091 0.241 0.134 1.396 0.090 0.862 0.026
    351 77587 SENSE 0.178 0.083 0.096 0.665 0.995 0.426 0.632 0.036
    353 77619 SENSE 0.389 0.058 0.398 0.011 1.913 0.257 0.857 0.040
    355 77629 SENSE 0.404 0.043 0.389 0.019 2.523 0.083 0.826 0.040
    336 77821 SENSE 0.393 0.063 0.444 0.033 0.196 0.228 0.619 0.015
    356 77832 SENSE 0.134 0.238 0.304 0.011 0.914 0.156 0.413 0.050
    352 77933 SENSE 0.112 0.044 0.165 0.009 0.749 0.482 0.517 0.171
    347 77580 SENSE −0.019 0.814 0.087 0.018 −0.158 0.151 0.124 0.204
  • If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 242, 255, 307, 313, 327, 330, 349, 366, or 387 showed improved salt stress tolerance evidenced by the second criteria as illustrated in Example 1L.
    • 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 at
    at day 11 at day 14 day 14 day 14
    PEP Construct Delta P- Delta P- Delta P- Delta P-
    SEQ ID ID Orientation mean value mean value mean value mean value
    199 12223 SENSE / / / / / / 0.353 0.024
    214 18456 SENSE 0.320 0.018 0.274 0.033 3.109 0.073 0.553 0.006
    302 70812 SENSE 0.195 0.078 0.078 0.296 4 0.000 0.363 0.016
    234 70950 SENSE 0.322 0.056 0.427 0.016 4 0.000 0.381 0.127
    239 70985 SENSE 0.270 0.066 0.221 0.005 4 0.000 0.372 0.007
    233 70987 SENSE 0.227 0.205 0.128 0.470 4 0.000 0.495 0.000
    228 70992 SENSE 0.369 0.002 0.370 0.013 4 0.000 0.574 0.027
    244 70995 SENSE 0.379 0.049 0.320 0.065 4 0.000 0.491 0.003
    208 72783 SENSE 0.154 0.084 0.043 0.553 4 0.000 0.264 0.044
    255 72807 SENSE 0.165 0.080 0.158 0.150 4 0.000 0.333 0.047
    256 72811 SENSE 0.489 0.052 0.491 0.050 4 0.000 0.784 0.016
    259 72825 SENSE 0.423 0.055 0.388 0.080 2.741 0.161 0.608 0.020
    295 72946 SENSE 0.345 0.053 0.473 0.050 0.898 0.632 0.415 0.181
    286 73026 SENSE 0.636 0.021 0.619 0.028 2.741 0.161 0.752 0.045
    290 73123 SENSE 0.460 0.048 0.445 0.007 NA NA 0.609 0.069
    278 73161 SENSE 0.510 0.024 0.506 0.006 2.624 0.246 0.626 0.073
    284 73165 SENSE 0.304 0.101 0.285 0.044 2.499 0.270 0.643 0.077
    293 73172 SENSE 0.197 0.056 0.137 0.204 2.374 0.293 0.660 0.081
    262 73242 SENSE 0.305 0.029 0.332 0.022 2.249 0.317 0.677 0.085
    265 73256 SENSE 0.325 0.084 0.182 0.178 2.124 0.341 0.694 0.089
    308 73485 SENSE 0.133 0.070 0.039 0.583 1.999 0.365 0.711 0.093
    291 73981 SENSE 0.239 0.147 0.238 0.281 1.874 0.389 0.728 0.096
    316 74566 SENSE 0.451 0.004 0.248 0.118 1.749 0.412 0.745 0.100
    312 74688 SENSE 0.190 0.191 0.102 0.520 1.624 0.436 0.762 0.104
    330 74863 SENSE 0.174 0.164 0.216 0.109 1.499 0.460 0.779 0.108
    366 77140 SENSE 0.644 0.005 0.572 0.015 1.374 0.484 0.796 0.112
    361 77150 SENSE 0.313 0.047 0.359 0.044 1.249 0.508 0.813 0.116
    365 77187 SENSE 0.273 0.084 0.032 0.763 1.124 0.532 0.830 0.120
    375 77195 SENSE 0.319 0.021 0.256 0.041 NA NA 0.847 0.124
    379 77203 SENSE 0.113 0.485 0.019 0.829 0.999 0.555 0.864 0.127
    377 77261 SENSE 0.115 0.230 0.085 0.315 0.874 0.579 0.881 0.131
    270 77308 SENSE 0.174 0.321 0.074 0.700 0.749 0.603 0.898 0.135
    346 77338 SENSE 0.227 0.158 0.196 0.272 0.624 0.627 0.915 0.139
    350 77357 SENSE 0.169 0.300 0.259 0.066 2.715 0.169 0.303 0.020
    390 77432 SENSE 0.306 0.043 0.122 0.270 2.570 0.214 0.734 0.019
    347 77580 SENSE 0.300 0.107 0.240 0.009 2.288 0.119 0.500 0.179
    353 77619 SENSE 0.142 0.216 0.139 0.002 4 0.000 0.129 0.582
    356 77832 SENSE 0.236 0.006 0.266 0.001 1.333 0.435 0.286 0.037
    343 77917 SENSE 0.174 0.163 0.160 0.006 4 0.000 0.330 0.009
  • If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference.
  • If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 209, 245, 260, 274, 282, 310, 321, 325, 336, 372, 376, 378, 380, or 382 showed improved osmotic stress tolerance evidenced by the second criteria 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 illustrated in Table 8.
  • TABLE 8
    Rosette area Rosette area Rosette area
    at day 8 at day 28 difference
    PEP Construct Nomination Delta Risk score Delta
    SEQ ID ID ID Orientation mean P-alue mean P-value mean P-value
    199 12223 CGPG1133 SENSE 0.112 0.617 0.334 0.014 0.273 0.143
    218 18231 CGPG3274 SENSE −0.131 0.269 1.144 0.002 1.179 0.003
    215 18414 CGPG3002 SENSE 0.997 0.034 1.665 0.001 1.895 0.001
    214 18456 CGPG2813 SENSE 0.099 0.757 0.799 0.001 0.684 0.001
    233 70987 CGPG4048 SENSE 0.864 0.136 1.213 0.010 1.146 0.002
    244 70995 CGPG4154 SENSE 1.088 0.008 1.262 0.023 1.407 0.025
    208 72783 CGPG2206 SENSE 0.814 0.023 0.957 0.018 0.899 0.052
    273 72788 CGPG5535 SENSE 0.262 0.058 1.090 0.016 1.212 0.021
    256 72811 CGPG4926 SENSE 0.880 0.058 0.669 0.002 0.715 0.018
    259 72825 CGPG5001 SENSE 0.032 0.887 0.338 0.071 0.357 0.027
    281 73033 CGPG5646 SENSE 0.322 0.394 1.311 0.012 1.480 0.017
    293 73172 CGPG5783 SENSE 0.395 0.209 1.065 0.030 1.099 0.074
    253 73330 CGPG4765 SENSE 0.347 0.515 0.835 0.065 1.043 0.007
    264 73735 CGPG5171 SENSE 0.624 0.076 0.655 0.001 0.718 0.005
    315 74460 CGPG6747 SENSE 0.452 0.005 0.542 0.043 0.455 0.154
    296 74746 CGPG5856 SENSE 0.304 0.346 0.978 0.001 1.061 0.007
    299 75240 CGPG5957 SENSE 0.479 0.153 0.652 0.143 0.875 0.039
    338 75654 CGPG7804 SENSE 0.531 0.140 1.712 0.010 1.979 0.015
    339 75692 CGPG7823 SENSE 0.526 0.063 2.027 0.001 2.246 0.001
    323 76161 CGPG7168 SENSE 0.768 0.000 1.810 0.004 2.146 0.003
    217 76532 CGPG3235 SENSE 0.921 0.008 1.216 0.001 1.324 0.002
    357 76802 CGPG8987 SENSE 0.468 0.097 1.578 0.000 1.872 0.000
    358 76829 CGPG9013 SENSE −0.434 0.329 0.709 0.010 0.314 0.118
    373 77121 CGPG9183 SENSE 0.081 0.385 0.757 0.023 0.698 0.014
    366 77140 CGPG9145 SENSE −0.221 0.165 0.896 0.007 1.043 0.005
    374 77157 CGPG9186 SENSE 0.190 0.566 1.110 0.016 1.176 0.015
    369 77165 CGPG9155 SENSE 1.573 0.043 0.683 0.038 0.699 0.022
    370 77166 CGPG9163 SENSE 0.777 0.011 1.432 0.016 1.570 0.025
    381 77204 CGPG9238 SENSE 0.545 0.139 1.313 0.003 1.436 0.006
    391 77433 CGPG9341 SENSE 1.054 0.103 0.895 0.027 0.659 0.084
    345 77570 CGPG8015 SENSE 0.538 0.215 0.866 0.025 0.898 0.023
    353 77619 CGPG8152 SENSE −0.589 0.322 0.503 0.068 0.558 0.049
    354 77621 CGPG8166 SENSE 0.053 0.863 0.716 0.044 0.792 0.040
    203 13411 CGPG1301 SENSE 0.183 0.427 0.577 0.033 0.573 0.031
    209 17210 CGPG2217 SENSE −0.244 0.685 0.162 0.038 0.156 0.104
    220 18256 CGPG3363 SENSE 0.272 0.400 0.639 0.008 0.719 0.020
    289 73134 CGPG5721 SENSE 0.014 0.947 1.053 0.000 1.062 0.025
    263 74217 CGPG5144 SENSE 0.168 0.390 0.261 0.043 0.206 0.075
    379 77203 CGPG9230 SENSE 0.606 0.051 0.620 0.032 0.778 0.039
  • If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance)
  • If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 255, 272, or 330 showed improved cold stress tolerance evidenced by the second criterial as illustrated in Example 1L.
    • 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 experiment 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/m2/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
    209 17210 CGPG2217 SENSE 0.164 0.089 4.000 0.000
    219 18232 CGPG3275 SENSE −0.081 0.613 4.000 0.000
    226 19767 CGPG3918 SENSE 0.212 0.007 4.000 0.000
    227 19774 CGPG3920 SENSE 0.140 0.011 4.000 0.000
    237 19947 CGPG4069 SENSE / / 4.000 0.000
    231 70201 CGPG3994 SENSE 0.311 0.010 4.000 0.000
    252 70684 CGPG4588 SENSE 0.335 0.209 4.000 0.000
    236 70915 CGPG4058 SENSE 0.231 0.258 4.000 0.000
    243 70956 CGPG4140 SENSE 0.113 0.525 4.000 0.000
    241 70963 CGPG4121 SENSE 0.210 0.252 4.000 0.000
    239 70985 CGPG4088 SENSE 0.283 0.063 4.000 0.000
    233 70987 CGPG4048 SENSE 0.392 0.202 4.000 0.000
    228 70992 CGPG3962 SENSE 0.008 0.959 4.000 0.000
    244 70995 CGPG4154 SENSE 0.426 0.002 4.000 0.000
    268 72001 CGPG5221 SENSE 0.070 0.728 4.000 0.000
    274 72753 CGPG5540 SENSE 0.189 0.010 2.889 0.121
    273 72788 CGPG5535 SENSE 0.548 0.009 4.000 0.000
    287 73120 CGPG5704 SENSE 0.401 0.146 4.000 0.000
    290 73123 CGPG5728 SENSE 0.171 0.166 4.000 0.000
    277 73137 CGPG5587 SENSE 0.382 0.019 4.000 0.000
    283 73141 CGPG5659 SENSE 0.348 0.029 1.891 0.219
    285 73155 CGPG5684 SENSE 0.194 0.105 4.000 0.000
    278 73161 CGPG5594 SENSE 0.327 0.018 2.889 0.121
    265 73256 CGPG5194 SENSE 0.176 0.473 4.000 0.000
    266 73260 CGPG5200 SENSE 0.256 0.047 4.000 0.000
    253 73330 CGPG4765 SENSE 0.229 0.016 4.000 0.000
    310 73411 CGPG6440 SENSE 0.232 0.213 4.000 0.000
    260 73628 CGPG5025 SENSE 0.304 0.040 1.330 0.467
    204 73944 CGPG1458 SENSE 0.426 0.026 4.000 0.000
    207 74065 CGPG1828 ANTI-SENSE 0.215 0.026 2.599 0.205
    248 74536 CGPG442 SENSE −0.054 0.431 4.000 0.000
    312 74688 CGPG6653 SENSE 0.645 0.006 4.000 0.000
    340 75657 CGPG7828 SENSE 0.394 0.017 2.680 0.180
    325 76178 CGPG7225 SENSE 0.334 0.107 4.000 0.000
    321 76460 CGPG7121 SENSE 0.136 0.381 4.000 0.000
    305 76527 CGPG6306 SENSE 0.238 0.001 0.335 0.421
    217 76532 CGPG3235 SENSE 0.250 0.331 4.000 0.000
    213 76602 CGPG2653 SENSE 0.283 0.387 4.000 0.000
    306 77020 CGPG6318 SENSE 0.507 0.038 0.000 0.000
    364 77139 CGPG9137 SENSE 0.445 0.005 0.000 0.000
    372 77180 CGPG9180 SENSE 0.370 0.046 4.000 0.000
    365 77187 CGPG9141 SENSE 0.456 0.036 4.000 0.000
    381 77204 CGPG9238 SENSE 0.314 0.002 4.000 0.000
    382 77266 CGPG9259 SENSE 0.017 0.948 4.000 0.000
    389 77430 CGPG9322 SENSE 0.095 0.233 4.000 0.000
    392 77444 CGPG9344 SENSE 0.171 0.292 4.000 0.000
    319 77517 CGPG6952 SENSE 0.132 0.001 2.507 0.235
    327 77536 CGPG7272 SENSE −0.084 0.675 4.000 0.000
    351 77587 CGPG8107 SENSE 0.415 0.112 4.000 0.000
    317 77610 CGPG6805 SENSE 0.267 0.180 4.000 0.000
    356 77832 CGPG8976 SENSE 0.153 0.579 4.000 0.000
  • If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 347 showed improved cold stress tolerance evidenced by the second criteria 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 may 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 may be associated with higher yields. Genes that favor growth under low light conditions may 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 ½MS 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/m2/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 Petiole
    Leaf angle weight at length at
    at day 23 day 23 day 23
    PEP Construct Nomination RS Delta Delta
    SEQ ID ID ID Orientation mean P-value mean P-value mean P-value
    251 70755 CGPG4473 SENSE NA NA −0.751 0.017 −0.307 0.059
    244 70995 CGPG4154 SENSE NA NA 0.251 0.070 0.008 0.944
    275 72709 CGPG5568 SENSE NA NA −0.903 0.070 −0.608 0.019
    210 72724 CGPG2292 SENSE NA NA −0.707 0.098 −0.638 0.020
    272 72774 CGPG5518 SENSE NA NA −0.268 0.110 −0.195 0.087
    258 72813 CGPG4977 SENSE NA NA −0.469 0.092 −0.186 0.054
    294 73020 CGPG5791 SENSE NA NA −1.443 0.010 −1.265 0.010
    288 73133 CGPG5714 SENSE NA NA −0.852 0.009 −0.896 0.012
    260 73628 CGPG5025 SENSE NA NA 0.311 0.011 0.317 0.146
    271 73766 CGPG5432 SENSE NA NA −0.988 0.048 −0.671 0.088
    314 74435 CGPG6737 SENSE NA NA −0.735 0.042 −0.755 0.049
    331 74858 CGPG7371 SENSE NA NA −0.667 0.010 −0.808 0.008
    330 74863 CGPG7316 SENSE NA NA 0.176 0.060 0.168 0.515
    332 74933 CGPG7457 SENSE NA NA −0.100 0.801 −0.533 0.094
    340 75657 CGPG7828 SENSE NA NA 0.193 0.059 0.244 0.085
    337 75685 CGPG7767 SENSE NA NA −0.917 0.075 −0.974 0.049
    267 75822 CGPG5210 SENSE NA NA −0.267 0.194 −0.297 0.090
    324 76171 CGPG7206 SENSE NA NA −0.350 0.065 −0.570 0.049
    254 76202 CGPG4788 SENSE NA NA −1.153 0.009 −1.713 0.019
    326 76467 CGPG7267 SENSE NA NA −0.484 0.177 −0.743 0.088
    359 76961 CGPG9080 SENSE NA NA −0.425 0.098 −0.265 0.051
    360 76973 CGPG9081 SENSE NA NA 0.333 0.052 0.248 0.003
    363 77103 CGPG9134 SENSE NA NA 0.363 0.093 0.198 0.072
    374 77157 CGPG9186 SENSE NA NA 0.287 0.044 0.107 0.086
    369 77165 CGPG9155 SENSE NA NA −0.081 0.413 −0.572 0.012
    368 77176 CGPG9148 SENSE NA NA −0.577 0.025 −0.651 0.085
    365 77187 CGPG9141 SENSE NA NA 0.335 0.002 0.263 0.046
    385 77209 CGPG9278 SENSE NA NA 0.671 0.027 0.451 0.011
    383 77220 CGPG9271 SENSE NA NA 0.463 0.008 0.231 0.228
    225 77334 CGPG3638 SENSE NA NA 0.531 0.067 0.336 0.031
    349 77349 CGPG8083 SENSE NA NA 0.133 0.001 0.035 0.781
    319 77517 CGPG6952 SENSE NA NA −0.843 0.142 −1.458 0.062
    320 77518 CGPG6953 SENSE NA NA 0.354 0.007 0.380 0.010
    307 77609 CGPG6326 SENSE NA NA 0.252 0.087 0.211 0.145
  • For “seeding weight” and “leaf angle”, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement 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 improvement as compared to the reference. If p<0.2 and delta <0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 246, 295, 303, 325, or 375 showed enhanced shade tolerance by the second criteria 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/m2/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 Root Seedling
    length at length at weight at
    day 10 day 14 day 14
    PEP Construct Nomination Delta P- Delta P- Delta P-
    SEQ ID ID ID Orientation mean value mean value mean value
    227 19774 CGPG3920 SENSE 0.488 0.097 0.307 0.100 0.550 0.163
    229 19956 CGPG3972 SENSE 0.248 0.062 0.154 0.214 0.284 0.001
    232 19973 CGPG4026 SENSE 0.326 0.047 0.066 0.577 0.444 0.012
    302 70812 CGPG607 SENSE 0.704 0.037 0.546 0.031 0.740 0.033
    236 70915 CGPG4058 SENSE 0.292 0.005 0.278 0.008 0.331 0.114
    230 70948 CGPG3990 SENSE 0.255 0.269 0.093 0.229 0.375 0.026
    234 70950 CGPG4052 SENSE 0.106 0.577 0.124 0.374 0.239 0.041
    235 70962 CGPG4057 SENSE 0.171 0.075 0.144 0.226 0.283 0.015
    241 70963 CGPG4121 SENSE 0.149 0.057 0.112 0.074 0.062 0.513
    242 70994 CGPG4122 SENSE 0.198 0.063 0.121 0.114 0.240 0.124
    244 70995 CGPG4154 SENSE 0.132 0.052 0.101 0.006 0.198 0.045
    249 71328 CGPG4454 SENSE 0.209 0.136 0.251 0.122 0.445 0.093
    268 72001 CGPG5221 SENSE 0.183 0.060 0.142 0.042 0.195 0.149
    274 72753 CGPG5540 SENSE 0.113 0.045 0.053 0.348 0.081 0.730
    282 73105 CGPG5656 SENSE 0.046 0.577 0.138 0.057 0.298 0.142
    292 73136 CGPG5764 SENSE 0.212 0.077 0.133 0.027 0.330 0.037
    265 73256 CGPG5194 SENSE 0.123 0.503 0.371 0.135 0.623 0.069
    313 74420 CGPG6712 SENSE 0.249 0.085 0.093 0.358 0.363 0.042
    316 74566 CGPG6796 SENSE 0.169 0.003 0.147 0.114 0.173 0.171
    304 74662 CGPG6185 SENSE −0.205  0.337 0.009 0.878 −0.009 0.958
    312 74688 CGPG6653 SENSE 0.221 0.083 0.059 0.358 0.295 0.163
    329 74862 CGPG7308 SENSE 0.201 0.122 0.044 0.515 0.383 0.006
    325 76178 CGPG7225 SENSE 0.217 0.018 0.093 0.364 0.240 0.234
    322 77069 CGPG7163 SENSE / / / / 0.641 0.074
    376 77124 CGPG9207 SENSE 0.337 0.121 0.256 0.045 0.762 0.034
    371 77155 CGPG9170 SENSE 0.181 0.262 0.265 0.035 0.419 0.000
    367 77164 CGPG9147 SENSE 0.589 0.068 0.369 0.066 0.899 0.051
    370 77166 CGPG9163 SENSE 0.383 0.025 0.391 0.006 0.787 0.019
    372 77180 CGPG9180 SENSE 0.442 0.058 0.267 0.031 0.402 0.339
    362 77186 CGPG9133 SENSE 0.448 0.022 0.383 0.079 0.654 0.057
    365 77187 CGPG9141 SENSE 0.453 0.043 0.227 0.127 0.660 0.013
    375 77195 CGPG9205 SENSE 0.221 0.114 0.188 0.160 0.259 0.064
    385 77209 CGPG9278 SENSE 0.360 0.092 0.181 0.203 0.594 0.047
    382 77266 CGPG9259 SENSE / / / / 0.470 0.020
    384 77268 CGPG9275 SENSE 0.353 0.111 0.170 0.244 0.386 0.041
    378 77273 CGPG9220 SENSE 0.255 0.070 0.023 0.586 0.510 0.141
    297 77312 CGPG5927 SENSE / / / / 1.006 0.065
    393 77409 CGPG9345 SENSE 0.410 0.027 0.396 0.011 0.461 0.040
    389 77430 CGPG9322 SENSE 0.209 0.183 0.211 0.247 0.399 0.092
    390 77432 CGPG9335 SENSE 0.342 0.113 0.285 0.122 0.425 0.055
    391 77433 CGPG9341 SENSE 0.204 0.120 0.222 0.041 0.382 0.085
    392 77444 CGPG9344 SENSE 0.185 0.068 0.227 0.003 0.276 0.002
    387 77451 CGPG9309 SENSE 0.266 0.097 0.208 0.042 0.014 0.981
    388 77452 CGPG9311 SENSE 0.286 0.021 0.182 0.123 0.284 0.201
    327 77536 CGPG7272 SENSE 0.121 0.389 0.147 0.026 0.384 0.027
    342 77549 CGPG7933 SENSE 0.349 0.006 0.207 0.032 0.383 0.022
    344 77568 CGPG8012 SENSE 0.312 0.020 0.157 0.015 0.359 0.071
    351 77587 CGPG8107 SENSE 0.151 0.128 0.223 0.014 0.370 0.030
    318 77618 CGPG6810 SENSE 0.179 0.018 0.104 0.276 0.154 0.388
    334 77816 CGPG7529 SENSE 0.145 0.400 0.171 0.142 0.312 0.181
    336 77821 CGPG7737 SENSE 0.114 0.406 0.126 0.208 0.311 0.079
    343 77917 CGPG7986 SENSE 0.136 0.083 0.162 0.011 −0.242 0.458
  • If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 252, 309, or 310 showed improved early plant growth and development evidenced by the second criteria 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/ft3. 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/m2/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 dry Rosette Seed net dry Silique dry Silique
    weight radius at weight weight length at
    at day 53 day 25 at day 62 at day 53 day 40
    PEP Construct Delta Delta Delta Delta Delta
    SEQ ID ID mean P-value mean P-value mean P-value mean P-value mean P-value
    394 12313 −0.075 0.489 0.654 0.000 0.157 0.174 −0.024 0.275
    221 18258 −0.494 0.017 −0.183 0.083 0.533 0.006 −0.269 0.234 0.056 0.009
    246 70657 0.373 0.040 0.187 0.037 0.355 0.081 −0.187 0.222 −0.003 0.972
    247 70660 −0.010 0.936 −0.072 0.431 0.445 0.026 0.116 0.369 0.145 0.045
    216 71538 −0.167 0.168 0.017 0.686 0.521 0.005 −0.196 0.113 0.023 0.557
    269 72056 0.563 0.008 NA NA −0.251 0.159 −0.289 0.314 −0.046 0.314
    279 73057 −0.266 0.035 0.167 0.114 0.396 0.034 −0.304 0.085 0.023 0.543
    311 73568 −0.140 0.115 −0.085 0.340 0.553 0.019 0.344 0.010 0.040 0.458
    222 19193 −0.296 0.110 0.081 0.385 1.076 0.011 NA NA 0.073 0.001
    262 73242 0.159 0.132 0.133 0.050 1.074 0.009 0.652 0.002 0.045 0.048
    309 73433 0.477 0.026 0.113 0.120 1.140 0.006 0.552 0.016 −0.009 0.872
    316 74566 −0.387 0.023 0.104 0.047 1.213 0.003 −0.169 0.130 0.012 0.598
    338 75654 0.604 0.012 −0.006 0.899 −1.193 0.004 −0.258 0.057 −0.004 0.962
    361 77150 0.611 0.003 −0.098 0.115 −0.363 0.011 −0.398 0.203 0.001 0.990
    327 77536 0.043 0.692 0.089 0.040 0.725 0.015 0.419 0.016 0.075 0.105
    334 77816 −0.324 0.021 0.005 0.954 1.207 0.000 −0.038 0.679 0.039 0.378
  • If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
    • K. Low 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 NH4NO3 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
    Root length Leaf color Rosette weight
    PEP Construct Nomination Delta Risk score Delta
    SEQ ID ID ID Orientation mean P-value mean P-value mean P-value
    200 10422 CGPG117 ANTI- −0.435 0.008 1.311 0.021 0.050 0.396
    SENSE
    206 12602 CGPG170 SENSE −0.004 0.941 −0.052 0.895 0.115 0.048
    202 13235 CGPG1288 SENSE −0.047 0.267 −0.085 0.864 0.128 0.068
    203 13411 CGPG1301 SENSE 0.323 0.202 −3.233 0.126 0.174 0.044
    201 13485 CGPG1226 ANTI- 0.311 0.096 1.162 0.038 0.241 0.019
    SENSE
    205 13846 CGPG1542 ANTI- −0.267 0.190 1.506 0.004 0.176 0.281
    SENSE
    212 16610 CGPG2499 SENSE −0.037 0.677 1.325 0.004 −0.004 0.918
    211 17805 CGPG2457 ANTI- −0.348 0.016 3.523 0.067 −0.105 0.116
    SENSE
    218 18231 CGPG3274 SENSE −0.207 0.117 0.925 0.062 −0.093 0.094
    224 18354 CGPG3534 SENSE 0.104 0.070 −0.407 0.124 0.189 0.048
    252 70684 CGPG4588 SENSE 0.379 0.001 −3.676 0.003 0.328 0.030
    223 71301 CGPG3528 SENSE −0.394 0.027 1.269 0.042 −0.128 0.026
    250 71329 CGPG4456 SENSE −0.169 0.230 0.475 0.074 −0.365 0.337
    258 72813 CGPG4977 SENSE −0.361 0.193 0.521 0.076 0.013 0.120
    309 73433 CGPG6429 SENSE 0.129 0.201 −0.091 0.794 0.186 0.002
    300 74349 CGPG5967 SENSE −0.139 0.019 0.592 0.080 0.045 0.080
    313 74420 CGPG6712 SENSE 0.612 0.043 −3.086 0.063 0.173 0.002
    298 75237 CGPG5941 SENSE −0.097 0.237 0.948 0.031 0.074 0.136
    333 75379 CGPG7520 SENSE 0.000 0.998 0.529 0.048 −0.017 0.755
    335 75434 CGPG7636 SENSE −0.336 0.039 1.208 0.076 −0.109 0.107
    341 75622 CGPG7833 SENSE −0.503 0.001 1.776 0.007 0.096 0.497
    339 75692 CGPG7823 SENSE −0.055 0.165 −0.377 0.328 0.132 0.083
    301 76422 CGPG6040 SENSE 0.197 0.178 −0.263 0.734 0.162 0.094
    359 76961 CGPG9080 SENSE −0.361 0.136 1.472 0.013 −0.190 0.066
    369 77165 CGPG9155 SENSE 0.366 0.041 −1.982 0.143 0.253 0.099
    303 77322 CGPG6178 SENSE / / 1.691 0.060 −0.185 0.052
    207 16322 CGPG1828 SENSE / / −0.021 0.982 0.116 0.043
    356 77832 CGPG8976 SENSE / / 2.472 0.011 −0.028 0.445
  • For leaf color and rosette weight, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p<0.2. For root length, if p<0.05, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2, the transgenic plants showed a trend of trait improvement as compared to the reference.
  • Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 198 or 327 showed improved tolerance to low nitrogen condition evidenced by the second criteria as illustrated in Example 1L and 1M.
    • 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 stage1.03 vs. not
    growth stage at day 14 salt stress tolerance screen 50% of plants reach stage1.03 vs. not
    growth stage at day 14 PEG induced osmotic stress tolerance 50% of plants reach stage1.03 vs. not
    screen
    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 −log2 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 events perform 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 day28 cold shock tolerance screen
    difference in rosette area cold shock tolerance screen
    from day 8 to day 28
    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 day14 Early plant growth and development screen
    Rosette dry weight at Late plant growth and development screen
    day 53
    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 log2 calculation. The Delta was calculated as log2M(transgenic)−log2M(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 log2 calculation. The Delta was calculated as log2M(transgenic)−log2M(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: 198 through SEQ ID NO: 394 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: 198 through SEQ ID NO: 394 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: 395 to SEQ ID NO: 19938. These orthologs are reported in Tables 2 as homologs to the proteins cognate to genes used in trait-improving recombinant DNA.
  • TABLE 2
    SEQ ID NO: homolog SEQ ID NOs
    198: 3331 2914 8408 6836 10845 14799 13562 8848 2509 13003 875 10945
    4176 7318 4820 19156 19137 14942 17166 13809 3506 3410 1343 8019
    7882 6490 3550 4999 3130 12635 8774 10864 17857 18089 16263 7295
    10882 18252 3728 14833 11485 15463 18826 2924 18723 1446 2812 7847
    3395 4209 15768 10679 18379 14040 3974 1087 11574 6329 5482 5609
    6441 5537 17042 4716 4516 12643 11384 12736 8137 1344 19473 8452
    4094 2460 18071 16891 5620 2364 7018 5200 11131 18133 12170 2731
    1687 5014 18511 9343 19283 6682 8123 8124 10597 10622 10608 10600
    16221 4280 11494 4867 9035 9507 7969 19159 19425 5248 17367 8135
    8773 1287 2671 9365 18729 13828 8754 7170 14529 16604 8341 1135
    6224 9739 11212 12962 4331 13555 14356 14357 14783 7509 2032 4692
    5243
    199: 15320
    200: 7710 14519 16336 7019 7872 7719 18299 10453 4650 15008 10089 699
    15645 10533 11751 12924 10526 5313 17532 8825 6941 7743 4155 14541
    9797 16905 8304 12080 19803 3087 1397 7534 5547 2423
    201: 11777 6444 1416 4353 15071 3413 14526
    202: 19073 6651 15740 7399 15791 2977 7789 9248 14697 15923 12131 5470
    965 11100 11069 12624 12892 3580 5270 18663 2916 1684 8107 4240
    19240 18582 19054 15775 7301 17416 10566 16351 13846 16386 18431 4439
    4056 8159 8292 12484 7316 3763 4783 18427 15501 16164 7762 4015
    4556 18955 12061 8636 9665 9713 10212 17555 5330 12850 16436 19045
    16034 16692 2571 11676 12666 11853 5348 8771 12218 3157 10749
    203: 5608 2382 17993 16598 7505 6976
    204: 11369 13527 10897 6893 6962 11592 837 5116 19669 1655 7244 2623
    2591 16504 17797 4636 15133 4141 7112 7939 6898 15965 16706 16979
    11784 16115 9620 15750 14652 3008 7838 14787 4563 1873 15811 7271
    4317 16603 1715 14288 11341 6188 1546 5683 5225 3033 6069 11268
    15981 15490 4181 8633 6058 7993 6763 9671 14892 17296 990 8184
    2935 8536 11232 12598 12638 5112 10787 1107 13858 5864 16398 16380
    17071 16520 1973 5455 13852 19228 1695 17402 2865 17120 2507 5601
    14952 1462 6616 6973 3092 1382 7755 3870 9765 5711 1257 6119
    15310 3792 15970 16032 14584 7282 15140 7273 3013 15249 13253 13241
    2265 17700 18976 4009 17354 2223 17559 3151 6320 17010 8939 11258
    734 573 15015 3050 16344 3931 11238 7691 11499 1112 4825 8344
    13519 11669 5640 13588 5473 15108 7210 17617 5994 5980 16627 17906
    5876 6079 18894 6956 12887 14694 10968 1061 2778 6096 10570 680
    974 3120 16300 5885 7626 13881 4244 4233 15039 4486 3103 3115
    7654 11890 12359 10800 9409 12657 8977 9182 17818 2942 9780 9899
    18496 4118 18188 10437 13907 17312 15730 13906 2603 12770 4837 12953
    16663 9237 9546 16293 18238 6022 13908 10266 9180 5538 15536 10370
    15958 5217 8154 13095 15972 14841 15262 12702 10766 15960 18726 19395
    10345 5964 13064 3946 17225 17262 17220 17266 17224 17252 5465 5495
    12800 12814 12758 3021 12400 12492 16461 13638 5616 17449 8397 17258
    5669 5675 6852 9796 176011 7569 9775 17664
    205: 14023 6117 12539 6696 18601 1463 19893 6621 9577 9166 17890 17889
    528 5413 18313 2784 8102 3988 890 19106 571 6791 2402 912
    206: 17912 8799 6566 12137 4356 5803 5253 6078 9123 17523 12392 17806
    6917 19039 7350 17767 11311 17082 15535 19392 11233 470 8318 14963
    16736 8867 14135 5246 4674 2124 9901 13302 15459 18997 2954 1026
    667 7561 16841 8092 9084 4065 1836 13713 13697 17640 7422 19580
    4565 15193 526 13108 7065 11703 11362 3633 19430 6221 17433 15616
    10082 10189 3089 15238 6633 8106 16180 15317 16364
    207: 14575 8831 11211 14985 17619 18829 2521 5219 15413 15835 1204 6569
    4661 4457 6066 14578 932 13694 7541 4813 15707 19563 1326 16107
    5009 7518 14825 13777 6175 15925 13617 5765 14838 9091 10776 15679
    5909 19373 756
    208: 19581 18655 16310 7497 6382 8261 936 7358 8883 4866 2146 8601
    2932 1626 10354 15192 11509 9909 13503 488 17477 14483 16941 2824
    1072 4989 14179 4883 13751
    209: 3493 16588 7031 5155 11150 17294 5241 10621 5786 5591 16509 2021
    1324 12454 15061 1670 15143 16506 17002 8741 18091 4013 5959 19370
    4239 12885 12174 3709 3431 9596 3378 1018 5963 13212 3150 2952
    1619 19861 19114 15446 19582 12663 19603
    210: 17777 843 11020 1337 18495 1493 6396 15336 14087 2568 4517 1157
    10741 4553 14999 425 4258 17388 5327 748 11691 10174 17805 16628
    10117 7980 8902
    211: 9637 17261 13232 7387 10506 19067 12021 872 11620 13603 841 1450
    2848 16911
    212: 14398 1558 18339 13428 13431 4182 16260 10619 6127 12005 7704 5064
    13072 4395 9032 19907 9588 478 12782 9917 13100 5202 10562 2044
    4452 10775 3543 14840 14071 14020 10406 14218 1797 16955 3879 10312
    12432 14824 7082 14554 15744 1508 15866 16232 6797 7077 5816 3597
    3596 3311 15022 881 2638 906 12037 12039 3458 2270 8812 3217
    9466 18102 610 9042 2431 18349 16650 14418 2251 4235 6826 1244
    16651 13004 7852 14435 9553 9262 15603 4340 9235 1413 10538 8296
    17223 16048 16071 2120 19847 10420 11239 18968 2995 7551 15854 12345
    5685 18180 2381 4814 2159 16862 8677 13466 11435 6649 6015 8097
    11230 16025 9440 1353 16151 16249 15930 11226 9398 2870 12373 15043
    11765 10145 11071 285 9270 9537 9095 2350 13362 5496 7924 11756
    2624 7967 6715 15074 17068 9275 10177 11007 14857 8951 12182 11643
    13590 7684 1740 929 910 9093 10018 13344 16978 17544 6305 11378
    1393 14108 15937 16631 11803 13126 11969 18524 9644 8425 8421 8420
    10755 10757 16696 15994 1143 3549 5868 5126 7506 12547 12669 19032
    4707 4710 4708 4732 4736 4733 8772 4730 4711 3385 1831 2528
    2304 15098 13223 15197 11673 4386 11884 2336 14693 9118 9633 14548
    12725 2399 18359 11011 14692 5652 5434 9755 9513 2681 4364 12903
    19777 13525 4326 6859 6088 11611 3068 13940 3578 14440 8582 9826
    19618 18012 17233 10057 18222 15021 9371 5985 14888 14001 1279 7826
    14232 18172 7425 11502 15565 17431 8317 10294 17742 5564 5531 18144
    7400 10835 14389 13461 13857 7113 16444 4722 2115 7276 18254 1237
    5256 13951 2920 16719 1045 15371 19022 684 8004 2781 19811 19766
    3279 18712 4940 14312 6426 10076 15772 7906 19836 11219 18985 2673
    3933 14263 11860 1907 4672 15599 13737 4944 2252 15415 11543 3433
    7746 17114 4115 4881 9445 14696 9525 14484 16112 5392 10428 1400
    9349 7616 18930 2533 12585 10497 10236 18415 4874 19793 4633 12544
    1995 17255 6130 10439 15261 6768 15985 17752 8504 12863 7487 14875
    12020 2036 18127 9264 3628 5019 7498 15865 1448 9636 2840 3161
    6207 2644 1059 2065 10031 2171 16927 4747 18701 19271 4174 1389
    9893 12527 9018 10035 5039 10698 13943 7894 12671 5651 19586 12810
    1254 3519 16114 4286 13948 14493 8294 14606 2586 14069 7712 16988
    737 16797 16826 18966 5145 17610 6304 8895 7744 9280 9284 10647
    16558
    213: 12662 5295 19093 10971 15359 1163 18513 889 2151 3536 7105 3292
    8466 18205 3579 1504 19486 13768 18047 6871 7174 11029 11505 17782
    5697 18822 4892 12656 1284 3062 4697 5993 18343 18790 3057 7014
    12265 10981 15204 11631 17704 17941 10633 19776 933 10363 8446 17800
    9822 2953 13693 19862 14551 15692 17673 6081 5049 12581 6910
    214: 15345 19484 4871 3871 6695 3993 11459 9199 15899 13821 14206 19489
    13765 712 19072 11649
    215: 1607 15669 13168 19044 790 9515 4511 13776 11573 13000
    216: 4929 17143 10058 17506 15064 3332 5268 6968 16252 15949 6180 3000
    14937 18454 13071
    217: 6807 19716 480 14021 4179 12236 6337 12237 6338 6336 12234 4815
    2261 18555 4706 7836 9176 18851 3443 9382 9439 8870 16691 16646
    11516 11500 11515 11498 16670 16660 16683 11513 11496 16668 16664 11519
    16686 11479 11481 7146 16643 16659 4850 17951 7144 4824 7163 4851
    11234 4860 17956 7160 17977 4827 4826 4859 7147 11475 7165 17928
    17958 4853 11473 7143 11477 5129 18893 18914 17526 17529 17548 17547
    17549 18917 13686 19089 13683 4938 16999 13666 4494 12074 17097 12093
    16874 12095 15878 17159 17154 17156 17153 5681 15126 10052 19712 4288
    15399 742 11607 15654 7006 8041 715 2713 11522 7695 3581 7865
    3776 15738 19821 17259 3948 13388 17427 15523 14216 8711 15423 19060
    16814 14298 18720 7885 3967 9710 3568 3573 3575 8503 15486 12932
    7870 989 3595 17756 9202 520 11205 14771 3574 2443 8036 19537
    14641 6739 15375 2400 6753 3336
    218: 13099 6217 4454 4699 11292 4702 1554 6467 15339 15722 11035 12964
    16317 13914 17486 18809 14651 4189 9581 14957 2871 1561 17618 3835
    14955 3868 9158 13663 10042 7575 19824 6200 10522 3600 4064 14590
    12731 7201 19572 9060 4035 6255 3457 11320 7986 8818
    219: 19325 10450 4487 12312 2564 10096 5641 1947 4367 6964 18031 1012
    11041 15078 12255 5761 5061 9832 16834 18348
    220: 8238 19207 18657 1358 2734 9320 13526 12631 17660 16548 15528 18501
    18049 15274 19470
    221: 2724 17089 9639 9208 17722 6817 11848 18904 13673 2347 11585 3054
    7486 6056 1290 14932 14930 12636 14230 1572 15708 18912 18177 1699
    10460 2090 5583 18802 606 5929 5041 19148 14224 9144 2643 17144
    14142 14674 16275
    222: 14820 18626 11591 649 3005 3037 5453 5430 14248 2371 4626 11661
    9784 10560 16219 11699
    223: 17739 13288 4139 15466 19773 753 1277 14333 4211 14510 662 7930
    18703 3268 19400 17533 2302 9078
    224: 13859 17816 12692 10417 7051 5444 12975 1611 7050 17105 5023 3397
    13149 14751 10564 7331 7329 11868 9210 9211 18860 18879 14568 17920
    14497 12723 19352 10587
    225: 11265 17061 11261 17058 11267 17064 8014 8033 8018 508 8035 8038
    8013 8017 3554 13411 12545 12528 11997 12566 11922 12520 11958 12525
    12522 12553 11961 11979 5030 3464 3910 18901 14601 14600 840 15664
    15668 15121 15142 15119 15686 15690 15657 15688 15146 15164 15716 15148
    15656 11191 13623 12732 5050 5029 5011 5055 5035 5036 5032 5081
    5058 5013 5078 5026 5059 5077 5008 5053 5076 11939 11976 12541
    2898 17207 5179 5115 13674 13423 16078 9689 11174 5252 17279 17277
    11177 1867 1865 1823 1844 1842 1793 1824 1820 10671 3139 1123
    5805 12934 12755 7504 1091 17904 17909 17926 17902 17938 17907 17929
    10598 17935 17933 5298 17980 10601 5383 14050 6585 11379 16583 17978
    17814 4838 17812 2633 3257 3189 774 12008 9988 14268 18392 12567
    14920 12980 11959 7682 7685 13235 4127 3740 3766 3770 3769 3795
    14966 457 8521 19464 4847 13386 5024 13387 13401 5957 6571 7932
    8369 10496 10492 10490 10495 10517 7943 4958 4956 4923 4986 4961
    4951 5007 4937 4934 4984 4933 4924 4921 4936 4979 4932 4981
    4988 18200 18196 7176 4739 5479 8351 7177 12886 9521 6464 17681
    14504 907 16793 1869 14509 8882 19727 8798 1197 3419 14168 12846
    15739 1115 14165 19715 492 8873 1119 497 1194 3251 1116 8876
    13982 12860 14430 451 14969 6102 4358 2650 2649 11790 12925 5882
    19696 9551 3867 19796 18933 18929 833 4908 14217 4852 4858 16064
    14208 10736 4886 14214 16062 14172 10735 14210 10720 1885 14187 14190
    14184 14207 14703 14707 14709 14706 14390 7658 7659 7661 2582 13794
    5204 17305 16541 8914 16959 17283 9043 3924 2696 8656 6648 2675
    18504 17311 6052 9425 9406 9410 9411 6051 9452 6033 15964 6267
    6031 8489 8463 8865 9427 6268 8490 8493 15602 15621 14616 15649
    15652 15606 11723 17247 9423 15595 15624 16148 2240 7428 11722 11363
    2225 2196 5480 15579 15626 16142 7413 5483 11741 16145 16162 11725
    5481 18072 10973 12451 12340 15511 12185 4348 12754 8722 14544 6171
    10732 9492 19795 12165 15082 6617 9613 739 19520 2497 14005 11128
    18795 14331 14329 14334 16439 12070 4053 3942 2387 2391 2394 755
    754 2950 752 2377 19819 17119 7118 1360 2273 11458 13758 11215
    11274 11229 13178 5110 16239 12884 18765 16637 19693 16831 17824 10810
    15395 15420 17470 10005 16397 16394 14711 14728 15503 4401 801 12244
    12292 12308 12219 9033 16763 14392 16804 16785 9566 16806 14409 14391
    16762 9027 9568 9029 16788 9009 16780 14410 16781 16782 16802 3330
    3357 3326 9001 3358 5789 14387 14412 3360 3356 1643 1805 14061
    15787 5632
    226: 19514 17912 10095 10091 10252 15927 5360 18385 12313 19719 12823 5182
    1491 8631 4666 2562 9358 10461 3952 19610 9255 7782 16516 5161
    6343 4894 5577 16734 1381 16378 14552 12707 11907 7651 10404 17767
    2417 11311 17082 17741 11253 11254 7473 13718 6269 3242 7085 5224
    8787 16268 19692 10769 8867 1434 16082 19530 14789 13615 18997 2954
    9175 8092 9082 1813 1815 1817 639 3883 15427 2035 4435 3601
    414 7005 8000 1812 1131 1455 6221 11231 10817 15601 16270 6118
    2193 17500 14186 9141 3449 3089 13886 15786 15238 2001 3945 14973
    16180 15317 18083 18695
    227: 17372 8093 5527 2439 13784 14768 10702 11535 7750 761 18735 5946
    17426 17988 10652 2928 18398 2009 9559 5094 12948 8522 1305 17785
    5852
    228: 2790 5299 15433 5501 18128 16466 14960 6564 8782 4397 19662 16008
    10829 19118 18086 3428 17943 10935 15806 8101 1184 4968 6059 10388
    229: 7539 3499 12437 19471 14158 11575 6399 15966 17187 13512 7688 2131
    10858 4169 19567 7834 7585 14073 3954 1991 12940 7756 8167 18994
    9449 15859 4341 17025 14373 12191 12982 983 13764 19774 16247 12665
    12664 2619 18875 7515 11785 2849 10913 10717 4653 11887 905 1641
    3563 6183 16765 17773 4166 4694 14328 11986 12084 1625 12695 9681
    14082 17341 15047 17498 3530 3531 7241 7972 4790 15643 14365
    230: 12430 9914 10470 14976 6139 18895 8072 18544 7257 6437 15066
    231: 7185 13859 12150 12692 5555 6319 14735 16754 3121 2208 14915 18531
    2991 7950 2419 4378 18922 4377 18705 17105 5023 3397 2203 5350
    895 18953 5090 9210 9211 14568 19723 12433 3877 17892 19476 11685
    9653 9066 16392 7989 5056 6545 19418
    232: 10845 14019 10546 14942 7302 8774 13992 10864 7129 12564 10723 2440
    19878 3172 18252 12532 9942 2129 18826 18723 17324 19791 8580 3395
    5915 9604 7572 16359 8047 3408 5482 12915 9969 16513 1344 16044
    19210 18748 11940 14402 19311 5620 2364 19283 5249 3646 17251 17844
    14459 5315 9268 16190 19143 15124 418 3076 16204 17414 18692 13164
    8191 10636 13894 3423 2842 1173 7663 13250 18208 14090 19680 15815
    4805 10584 1287 15495 9256 2229 10166 19534 11163 2292 2687 19082
    9365 2465 5381 18660 3073 9545 17750
    233: 19344 12031 1229 15123 14123 19501 7767 5944 11173 4596 11614 10841
    14129 2393 7360 621 14382 11612 8136 14668 7577 1652 2491 3392
    2160 4767 17696 6615 12377 3471 2985 17194 17177 10988 5084 9695
    16519 11445 3213 10846 16453 19346 18274 17478 17798 15663 6823 5131
    14272 3777 19911 19724 6132 19722 14803 17148 1605 11787 14359 15796
    645 10159 13264 12931 6357 4621 16774 18913 5746 7161
    234: 11849 7119 1272 13625 6244 7780 7229 640 15779 14967 5388 14480
    1349 17525 12768 10327 8976 5113 5895 15006 4428 16619 4613 3393
    18954 4451 3799 17712 12488 15863 18697 18482 7844
    235: 8664 9584 8904 18497 19265 8237 2617 7729 17728 8659 13842 11016
    9197 10827 5351 3224 7379
    236: 12797 7459 12082 1618 7740 16856 7623 8994 7602 19549 12596 7644
    18396 13883 7266 18758 3328 18998 1660 12434 2139 19899 12252 5883
    3266 19587 11474 5870 5443
    237: 14746 16460 7754 9978 16450 18053 18050 18466 8866 10337 8245 1604
    8568 13606 19627 18283 15611 10648 13871 9910 10297 3025 18486 8908
    14474 11654 2349 10867 14488 13901 7336 11511 14836 1906 13400 5568
    19527 4351 18319 6689 10519 15977 15760 7605 16652 14204 14665 727
    9225 12804 2776 11108 14258 1882 4357 1972 8324 6435 18239 6728
    18983 9555 13551 10797 18458 4622 14064 2771 1818 18410 13553 15912
    10854 9475 10873 15530 14816 6283 703 8105 16216 7038 957 11908
    13853 10718 11237 5623 10262 12248 6541 7053 4480 14011 17142 6691
    7795 17981 2822 1473 3561 4545 13347 1348 11256 6511 11356 13955
    6699 6555 6559 3185 16166 17894 9307 6011 10635 1391 13177 16458
    702 18335 18740 16787 4175 10950 3412 4193 951 13870 19183 18644
    947 16280 6184 13111 3667 10338 4125 14782 18479 18004 19084 12471
    16645 866 1895 10049 11122 12521 7132 2660 12759 14534 18429 15615
    17611 9347 12704 1525 7492 3451 18373 17125 5972 9777 16039 12350
    4073 8962 17221 1961 1958 11156 12899 3908 7429 10977 15040 16234
    17004 17293 13355 4977 260 10645 14133 9251 12220 12633 7087 9535
    8894 11452 2346 6125 2062 6710 13299 12947 12943 4422 5817 13502
    14378 13504 14379 4770 1451 19815 12842 4405 13837 9184 10051 10999
    9565 12305 11330 16546 17073 1402 5263 7189 6160 2372 6567 1248
    3858 14455 1766 2516 1656 14550 16864 14989 14368 15573 9312 7786
    13516 2969 1952 2541 11207 10625 9198 19674 10334 18065 3495 8519
    12201 3921 13249 18592 857 17545 11757 15455 2164 789 4350 3117
    8649 11708 11463
    238: 9324 12427 663 6064 9836 19556 9348 1900 17219 13657 7086 7381
    6006 7357 10469 6466 8472 10588 17047 609 1637 10699 6450 14299
    15101 10536 18759 2378 18013 9269 3343 12489 11305 18178 2269 16890
    10629 18665 15962 9375 9437 16312 6501 11737 6339 10847 5290 4667
    5522 18388 1804 11566 2369 15872 12465 7687 6209 11354 15667 15938
    18971 18258 3774
    239: 13622 9907 10802 1635 19607 8218 11862 18989 19820 1430 15998 4855
    18040 3455 2471 6506 7587 11932 17440 16155 13643 12011 13012 11289
    14270 17959 10271 5763 19020 5904 7809
    240: 19167 14809 6116 11171 10424 1445 13848 1387 18366 9887 3732 10953
    2175
    241: 939 3338 2461 7962 16158 15297 19138 7398 3035 1767 9983 16494
    10256 11165 13953 18376 4113 4116 10457 11995 19241 2063 19340 15435
    14028 14611 14366 18488 9171 5494 10438 18123 11822 13093 15526 9516
    18346 4375 8716
    242: 12077 11851 16904 8919 18979 6443 17768 3571 5105
    243: 12862 6311 17138 19420 12939 8147 18538 16023 6580 8709 16080 4700
    2940 9554 5556 503 12303 7072 8226 11973 19415 12551 9852 13704
    7982 1030 14887 12346 1435 1437 6242 16606 2041 5356 12706 9139
    2872 10869 9028 7957 7190 19892 16505 9383 6589 11472 8394 8396
    3626 16568 19220 18648 14425 15305 13545 8443 8427 1247 17078 14914
    5103 19922 14358 18892 8972 16582 12124 19870 9538 19845 19867 19195
    19221 19191 19323 19242 19215 19307 19275 19273 19356 19192 19173 19350
    19279 19216 19852 19223 19250 19326 19848 19330 19214 19869 19351 19332
    19276 19247 19177 19163 19305 19328 19851 19353 19197 19850 19358 19175
    11034 19871 6578 17445 3832 3831 6233 7090 9456 6970 13917 13920
    4539 4537 4542 4028
    244: 1245 4324 12202 4379 6967 9036 9880 10540 13263 13138 18122 19047
    2008 19483 2468 7439 5787 10979 15035 10982 3699 11097 14482 17829
    11866 4198 14130 17239 6111 14956 17492 11385 4004 10448 9464 9894
    15398 1231 12878 12829 3833 2080
    245: 11361 5661 8686 17264 8328 8330 7550 10232 18956 315 8720 1696
    5922 16632 14340 11996 8012 4522 16791 10038 5066 4969 4634 17213
    14401 7284 2099 6718 8030 14321 1903 15554 15028 12204 3060 19178
    11653 9316 17858 18132 18589 9667 17297 5418 17508 15450 6147 5012
    18588 18020 862 4920 15050 16525 3064 2884 5607 12297 11396 18052
    2912 17060 1697 17604 18870 1732 5968 18761 4044 5088 10593 5242
    4527 13682 8054 16503 1962 19380 1623 10664 4597 15319 3478 16945
    18584 3108 12724 15546 15764 15754 18354 18416 4890 19678 19130 15532
    5213 11374 9753 14702 15217 15921 5855 18752 1904 15721 18203 18156
    18864 8700 7560 16924 636 6811 16379 19046 11063 14929 16207 15425
    17368 11052 14975 6574 9560 10349 3830 8250 2015 18785 8196 7713
    11586 14490 18301 18792 15378 7699 18803 19816 16104 14468 17612 11556
    1765 5525 2247 5751 11815 17200 10136 6205 16567 10487 18141 6202
    8537 8518 7037 8133 6764 827 4941 818 8356 19365 7588 3017
    6400 5637 8416 12444 510 12026 11634 9859 1460 18194 11314 2685
    4226 984 19513 8932 12905 4112 18873
    246: 13549 2112 6794 8833 12086 2984 5421 7868 9325 19092 9699 19026
    1631 4555 804 8588 19422 8685 6218 17573 17039 12147 4754 10543
    6834 6733 864 9209 4190 9301 16571 2360 4996 8081 11140 13006
    10637 5281 17394 4637 9663 1828 12569 1215 14407 13300 6644 11688
    17465 631 16880 3874 16141 975 8011 16154 18389 13579 15094 8944
    3735 8021 2288 15334 16083 15801 12760 10618 13208 19889 18999 4870
    13710 12132 15259 9267 11470 13144 17588 628 17272 14844 14000 19900
    15096 12603 13869 6532 16013 5431 1192 10395
    247: 14941 2202 18124 13094 7945 3968 8551 2458 18176 15639 12789 5427
    16829 9519 1674 19789 16819 4712 18517 13990 16185 6271 15089 5575
    1227 1331 10634 4978 6366 14209 9402 12738 589 6685 16243 2070
    14026 12494
    248: 4050 14993 16855 19797 5528 9097 18611 19631 18510 8376 8956 10289
    11370 13805 18078 19606 8202 15826 16478 19041 5700 6765 8134 14325
    10101 16816 4765 16287 5377 18675 7230 7964 876 9597 19802 11403
    4833 17530 18265 13417 12779 8885 11923 18564 3790 19147 1406 7066
    10573 7108 3783 4727 19785 1458 16375 8415 9522 10729 6071 9047
    9113 3248 6378 1799 14779 3630 4294 7608 13707 16321 15368 12743
    11299 2413 1584 11214 4012 2710 4268 19546 17625 6940 1273 5755
    413 5952 3098 15675 11098 3286 8068 2690 1222 13381 15257 4756
    4725 9351 7586 5770 10868 3420 12012 775 7742 8422 6103 15004
    16010 12445 13845 17033 16212 3842 19401 4100 8417 13255 1233 5307
    7664 11820 4960 4282 2333 17315 12161 4217 10485 8151 16203 1002
    11057 5823 9298 1567 411 5686 2787 6556 17045 5742 3090 12019
    12930 14231 15548 11694 8266 16248 1579 4806 2747 11349 6186 8303
    4499 14141 8094 16001 12109 10916 10761 2142 4592 5947 1880 5294
    16111 2594 9869 6575 13083 17382 14364 11324 2341 14545 1596 15428
    18963 1929 10976 3736 16709 5662 4501 19083 17865 14066 8045 18782
    2866 19741 12401 690 3507 2118 8721 10292 3096 7017 5861 9569
    15479 6800 14522 15996 3926 12508 9266 7508 3882 1770 18129 2452
    16753 7303 5038 17638 14421 11636 19188 2092 5721 781 700 15887
    8872 5320 5466 10259 7441 13041 10874 7249 4514 9725 18158 17609
    18140 18293 17059 18509 4888 9306 5484 7091 1826 19360 3124 1056
    2563 3177 16370 10946 6177 11856 17835 15766 9377 14858 9666 2881
    4713 18553 10081 3260 6030 16206 11892 10319 18672 3859 2663 15443
    5089 10992 2411 12439 17434 6053 18213 1552 16896 16303 2170 4062
    9729 5506 4195 2496 9435 18250 3643 10322 8871 11520 8307 10814
    4172 3616 467 2220 2765 10994 7181 9294 9019 14302 16474 5797
    4407 12612 9186 18928 4415 11982 14037 3959 1335 507 14948 9483
    17387 6806 8933 14886 13678 506 12462 6785 2667 19322 5005 14242
    11045 12403 2390 19573 11726 9949 5099 9853 16159 15920 13256 615
    3439 10834 8240 12330 19303 17872 12750 7494 2539 12602 5373 16910
    3341 11873 10613 7321 18101 12158 14798 8096 5837 1986 13521 5384
    7186 4164 12183 5907 11172 15553 726 4059 6816 6374 16761 7732
    6913 1868 15780 18634 16779 6640 3707 4959 10722 8632 18898
    249: 8837 9354 459 481 15638 15637 10951 1014 1011 10934 460 456
    1750 19841 3504 7770 7490 2370 12273 8627 10651 13584 13749 13618
    14339 12485 7233 18749 12207 7057 408 14300 5215 19033 14163 1226
    1228 5775 5424 8505 18275 18314 10203 8674 11707 15070 19005 8386
    2693 9051 4549 8477 13649 15215 13783 10149 13027 13294 15862 674
    9926 18724 12578 8169 1419 6285 15077 2147 14574 6516 1177 13510
    9181 2520 4618 7286 7290 7135 6455 7288 9787 11002 551 2679
    8942 1701 8928 8930 1097 17269 12778 15439 17593 12347 1453 10948
    15909 19108 8061 16885 8964 5642 15311 18841 8881 9067 2902 10703
    8544 18226 18698 18197 7766 17357 6778 13002 10083 15189 7944 1067
    13701 18247 5067 8858 2375 10780 12562 6618 1888 368 13569 16443
    14088 17735 6201 1162 9317 18221 17786 9563 9562 2386 3903 5156
    18001 547 12480 13049 2495 8339 18977 12208 19466 10488 15681 11974
    4385 19012 8291 16077 16024 5878 19014 8302 17408 16026 16018 16022
    4841 9829 9828 17831 4690 2646 9830 13874 14131 9842 9827 9849
    8403 9844 9847 9865 9851 8406 8404 6995 7012 8405 4019 10234
    7448 17662 1677 19490 2475 6216 13658 5135 4005 982 15959 17990
    3508 17774 17778 15478 7855 9687 17808 10782 7592 268 18627 12803
    17776 12927
    250: 12939 8147 18538 6832 13653 16376 2940 8025 9554 5556 503 12303
    11973 12551 9852 1030 10923 12346 1435 1437 2658 15612 16349 9028
    13226 19568 6589 11472 4670 17175 1302 15214 13545 992 5103 19327
    18189 7434 13340 9873 7090 16545 6970 15069 19166 16647
    251: 6603 10030 13559 6740 11390 7765 3614 11615 15027 11616 14213 11329
    19168 2567 13201 10961 3308 11492 2325 6423 11527 3741 11560 8630
    7463 1786 14408 10585 15696 14167
    252: 17378 17392 17375 19787 819 9037 18778 15271 2631 2615 2616 2606
    15288 2607 15269 15283 15286 15282 15287 15285 2609 2629 2613 2604
    1472 3730 6707 4595
    253: 16186 14031 2169 19019 14212 16029 16170 16973 2492 10956 13426
    254: 9072 16169 1130 12933 1592 17856 19381 14964 19031 3682
    255: 15936 5121 14535 14860 1527 1041 997 4031 18021 12960 12659 6385
    17513 15910 6705 15441 15200 3272 16975 18974 12341 5796
    256: 15904 2266 1296 6085 10365 4818 7630 6936 18652 4715 9134 15714
    2506 5391 14801 12410 18804 2831 19264 7856 15547 14814 17301 17179
    18891 11410 1475 11170 7067 17441 12196
    257: 1879 1595 12797 7459 8363 12502 12756 5854 465 1425 1893 12362
    12596 7644 16328 15689 4599 17970 5443 17245 10708 17790
    258: 3828 1761 11325 17218 18307 14003 12997 4456 6236 19270 8499 7402
    5087 10067 3938 3953 3939 17913 19475 5664 3918 12870 8678 9700
    259: 12266 4307 12661 6842 6841 6413 17238 16962 15778 516 7796 7046
    7155 8841 7260 9891 13142 2868 8814 9110 9530 14169 17860 3971
    6657 13557 14715 9995 14954 9514 14428 5521 11000 8008 6231 6796
    6894 12969 15894 13220 469 19780 17022 2260 17210 1668 1624 17554
    9991 2888 10752 8717 1792 7940 17358 7238 580 582 583 2828
    4970 4995 18273 1838 4998 4967 8488 14536 1477 13690 10891 14229
    7730 15048 9774 18163 9481 10644 2992 6652 4943 7469 6637 19611
    6346 6577 5487 15216 8436 7531 19737 9498 7963 8585 17924 10815
    10055 9630 14139 8171 9921 12928 12473 13070 793 5190 2500 9328
    1480 13432 15272 14189 13728
    260: 14746 16460 18053 7754 9978 16450 18050 18466 8866 10337 10391 8245
    1604 8568 8843 13606 19627 18283 15825 15808 15611 13871 2627 2601
    19077 1485 5898 5916 8835 2570 9910 10297 3025 18486 8908 14474
    11654 2349 10867 14488 9940 13901 7336 11511 19746 14836 1906 13400
    19527 5568 4351 18319 6689 10519 15977 15760 7605 16652 14204 14665
    2776 9225 1972 12804 727 14258 18708 1882 6435 11108 8324 18239
    3913 10328 4357 12240 6728 18983 18808 5969 9555 13551 18458 4622
    13553 10797 1818 14064 18410 6283 10854 2771 9475 10873 15912 15530
    8946 16216 7038 957 13243 13853 11908 12331 10718 11237 5623 10262
    12248 6541 17142 7053 14011 4480 2822 17981 7795 6691 3561 1473
    17141 4545 13347 1348 11256 6511 11356 8651 13955 6699 6555 16166
    6559 3185 17894 9307 6011 10635 1391 13177 702 16458 18740 16787
    4175 10950 3412 4193 951 13870 19183 18644 947 18835 6184 13111
    3667 10338 4125 14782 18479 18004 19084 12471 16645 866 1895 10049
    11122 12521 7132 14534 2660 12759 18429 17611 15615 12704 1525 9347
    7492 3451 18373 17125 5972 9777 16039 12350 4073 8962 17221 1961
    1958 11156 12899 18769 11606 3225 3930 12985 3908 7429 18285 10977
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    4569 6686 7903 18104 7660 11455 7569 10308 14385 17379 16758
    283: 15531 6256 3677 12101 13037 10190 10248 11043 822 16817 7460 8374
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    9599 10039 5643 1398 5309 2941 13830 626 17116 19140 11467 18598
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    17610 6304 8895 7744 9280 9284 10647 16558
    286: 9313 1653 17351 14049 14657 1383 1015 18068 4392 10779 16383 9920
    487 9934 2830 3247 2351 18430 10228 17404 17658 5336 5337 17655
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    8500 19667 4212 4448 3986 12515 15430 7679 18854 16043 4242 10959
    13366 2878 11272 5100 3297 7169 19051 3475 13999 17136 17946 7102
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    17653 13145 16135 7307 4655 9544 12992 17516 2232 1408 2230 1599
    11103 9661 8918 17531 7259 10941 16332 19728 2216 11076 2670 18069
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    18745 3786 17936 5887 3425 5214 8401 14931 18934 15251 6533 4671
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    10229 14451 7419 9477 11295 15793 15660 11783 701 2955 17659 2504
    17348 16342 15186 6406 8247 5380 13891 1315 1256 19915 1724 2012
    14065 10299 8282 616 13375 9102 16056 11991 9014 9223 19193 5659
    12275 17960 6092 13451 8592 8527 7016 15734 1394 3654 2224 9086
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    5846 5825 13392 3964 18837 3676 6815 1563 14197 4983 16851 1080
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    7627 18249 9111 10610 1083 6509 17564 5851 15935 14750 1415 7919
    8561 13034 1757 7298 10482 17985 2674 18011 18159 6510 8905 4745
    13968 5850 6365 18717 6735 7063 6097 7741 6692 16850 9791 1250
    1976
    287: 14033 3069 10887 17423 15804 10725 1039 8823 13656 1864 18512 3164
    11788 9149 8808 5438 15880 3992 800 12872 18261 8766 14438 3187
    10085 4070 901 13535 2677 8285 10764 19705 18563 18309 4608 19842
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    5003 15379 17113 11587 10350 16113 1863 10534 16794 10418 3427 14528
    9660 19113 14120 18931 693 10649 15876 620 11764 4473 14614 15160
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    7528 17188 5982 10710 4743 17869 13867 15895 18341 13127 9124 11340
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    11869 2959 17628 2207 1422 5187 12698 16068 18921 3969 18923 15856
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    18840 18022 3726 3772 13530 12761 10851 9602 19087 12685 5286 4627
    16074 10376 1178 17450 2560 825 10966 6542 5902 17622 13520 16539
    494 18321 9756 4078 13338 11983 15481 17256 1850 10700 6895 19331
    5603 16542 8629 14464 8541 11955 15582 1615 9393 11799 9705 10217
    3894 5124 17122 10037 19916 3503 17417 13729 5423 16076 1843 12859
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    12512 15134 8390 16883 10879 8150 9719 12593 14926 14282 17273 6293
    6538 13207 13438 1179 13854 1534 12605 12235 11858 6290 13936 15363
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    18036 14808 13308 6822 19066 15323 12540 9754 11217 12299 6562 19219
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    4965 17040 2183 16534 15799 9967 13632 6850 19691 8788 16409 4673
    17193 14098 11916 2451 4834 18460 18183 14691 19318 1489 12987 16441
    17707 16821 18525 6452 5795 10306 808 5051 9434 4528 13359 1168
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    6492 16565 17743 5954 16843 954 5541 5859 18096 12827 15357 11338
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    3632 1407 632 5735 2700 8621 4284 18605 5544 3482 7892 18630
    6701 13975 18530 11623 10209 10341 12178 19162 4832 13434 19639 7936
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    7891 10920 8887 5093 14119 434 10475 8949 9491 15868 17642 9386
    8713 17515 11664 12640 4630 9414 7285 9296 14419 9418 14870 5136
    2943 8767 9080 9232 9233 9981 8666 11032 6927 1544 17476 16917
    19548 12188 2244 15586 11795 14909 4751 13962 9106 429 18945 4616
    5199 10178 18224 7863
    288: 14033 3069 10887 17423 15804 10725 1039 8823 287 9557 13656 1864
    18512 3164 11788 9149 8808 5438 15880 3992 800 12872 18261 8766
    14438 3187 10085 4070 901 13535 2677 8285 10764 19705 18563 18309
    4608 19842 15814 13706 2652 1108 2600 11436 8221 18056 9465 14769
    5688 5397 3900 1598 6393 5004 17760 15162 12125 12647 7151 11629
    14327 14070 16315 15318 15939 1024 17173 7378 11794 17214 14034 2307
    3146 11136 745 14629 1219 8349 9131 14106 2574 6164 13786 12212
    5003 15379 17113 11587 10350 16113 1863 10534 16794 10418 3427 14528
    9660 19113 14120 18931 693 10649 15876 620 11764 4473 14614 15160
    2527 7562 9412 15931 15942 10452 1278 9417 14663 6750 3059 18739
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    6395 2512 1364 4248 9916 17140 19660 5811 9460 2383 2545 10421
    10451 7528 17188 5982 10710 4743 17869 13867 15895 18341 13127 9124
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    18536 11869 2959 17628 2207 1422 5187 12698 16068 18923 3969 18921
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    8268 18840 18022 3726 3772 13530 12761 10851 9602 19087 12685 5286
    4627 16074 10376 1178 17450 2560 825 10966 6542 5902 17622 13520
    16539 494 18321 9756 4078 13338 11983 15481 17256 1850 10700 6895
    19331 5603 16542 8629 14464 8541 11955 15582 1615 9393 11799 9705
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    19219 2211 1980 409 1970 1424 14183 3223 14118 17192 13802 12957
    12155 11050 12753 8961 6911 12324 7373 14643 15307 11544 6662 13247
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    9472 12965 12360 3202 11429 11427 19689 1587 773 8864 14046 5266
    9201 10510 17227 16534 7291 9967 10904 10655 19691 8788 16409 4673
    14098 2451 17193 11916 14691 4834 18460 12987 18183 2944 1489 19318
    17707 10306 5795 6452 13359 16821 18525 16441 808 5051 4528 9434
    1168 13904 954 5541 5859 17743 14884 5954 16843 18096 12827 15357
    6492 16565 11338 19639 5834 10246 3697 7974 4376 19409 11058 16197
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    10475 8949 9491 15868 17642 9386 8713 17515 11664 12640 4630 9414
    7285 9296 14419 9418 14870 5136 2943 8767 9080 9232 9233 9981
    8666 11032 6927 1544 16917 19548 12188 2244 15586 11795 14909 4751
    13962 9106 429 18945 4616 5199 10178 18224 7863
    289: 5523 4313 17448 17841 12777 10355 19605 19897 13150 13788 8075 5624
    13087 8533 15156 16282 4418 5362 17314 16470 9691 6830 3539 8947
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    6105 15005 17775 7340 16006 1428 19624 11919 12601 642 3843 3912
    4096 12974 13254 2664 16161 8861 13261 13575 9768 14949 19126 4880
    5824 9299 14874 5687 2782 17505 17726 6275 3175 13677 4398 4025
    5376 4677 18843 7388 15255 14128 2596 1938 14569 13062 4742 13091
    16870 9122 11112 12762 3052 11166 2182 15922 13893 6314 10549 17230
    10781 6932 11582 16011 6946 9000 6251 9407 11924 9877 6838 15556
    15794 11808 19431 2295 1090 12514 7454 18403 2529 18862 19419 12173
    3773 11501 1975 14174 16124 8277 11572 2186 4994 16607 12215 7089
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    15224 9191 8757 16205 13252 3135 18125 1294 2016 407 744 18246
    19479 15883 18721 4036 14388 15680 19029 5232 12705 8217 16267 8222
    11264 961 12449 16916 13761 3615 7172 14831 14301 16476 14882 7390
    14607 14441 5660 11599 11359 4092 17343 13568 581 5071 859 5934
    10571 12380 8016 725 9697 6714 8705 17074 1644 11944 12826 11957
    4502 5499 5549 14898 1200 7101 18165 6291 6133 13712 11143 13419
    3259 14157 11554 16748 6359 2357 941 11901 18451 1984 931 4926
    6943 18727 6551 17793 4278 4905 3893 11593 10509 15596 4274 3302
    3510 8231 18704 13258 4695 1347 8641 4776 16869 9696 16749 12938
    4466 5831 2176 13023 7184 5489 8886 12920 6529 871 15574 8579
    17493 7527 18633 2515 3708 13536 7991 3379 16897
    290: 13013 2572 15390 1939 14078 15522 9968 9379 18412 18308 9944 6870
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    4089 17112 14261 13039 2448 19588 19632 10171 3875 16215 6248 6904
    17027 14757 14672 18336 6783 16361 13415 7175 19494 2544 3982 12302
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    10809 10295 3281 8912 10017 11633 16868 2259 19421 16353 2746 14045
    11766 14772 5191 8574 17749 1617 17592 15384
    291: 17667 17054 19440 15213 7334 2141 3727 9845 11081 9526 12337 15918
    15597 3255 10276 970 16639 19309 2222 15291 19583 14945 7813 9966
    1286 1910 10491 19293 16030 19104 463 11758 9810 14676 9957 4241
    14669
    292: 9221 9220 6193 3253 14842 13721 4160 4741 7322 10303 16057 13497
    13494 13514 3142 3155 3203 3201 3204 3231 3227 3230 3228 5987
    3206 17127 17830 17527 19410 19413 12404 13866 13360 13864 13860 1092
    18317 16638 3344 11672 1048 5311 11768 18957 9486 5520 4119 3271
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    9650 10687 844 17322 17326 6887 4652 3798 14561 15275 12003 17738
    3837 3822 3819 3846 15647 8778 10880 17001 8637 3841 8776 18718
    14843 3863 3817 13443 13421 13442 13498 3923 13787 3280 3276 18190
    4216 12478 8586 7030 8614
    293: 13730 7076 16121 2661 2089 15753 13834 1549 18755 5880 14792 946
    10952 15392 19376 15572 11083 1945 12301
    294: 19341 4724 19896 3258 5111 599 6431 13589 6942 6192 6252 13509
    19882 4075 1265 10657 10589 5296 15003 18223 18248 2327 1261 9965
    4027 15223 16926 14122 18287 13182 19642 13929 3304 730 11185 4607
    2628 9008 18151 2691 11457 19048 6876 6857 15174 15158 17254 6991
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    12614 8990 7650 19393 5863 3904 10368 7157 16438 13128 17560 7535
    1575 10105 10134 568 13578 4304 9818 7538 9257 18048 15120 18444
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    342: 3006 14845 848 12847 17916 1499 5698 15349 8481 2524 12033 2479
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    343: 6892 17589 9778 16830 14586 3829 7946 7552 7372 2949 6234 6869
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    1891
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    357: 18266 7571 19462 4882 3947 18198 19775 19771 19267 9864 15678 7599
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    13648 16385 17968 17992 18014 17987 17989 17964 17984 17963 18015 17961
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    3775 3751 3778 3747 17028
    370: 13195 17260 15550 15298 16070 13528 18906 6667 3884 19731 16485 15332
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    17456
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    9121 5888 18291 17740 7117 12383 15552 8043 8998 8613 9367 10541
    17801 8616 10220 1388 8599 4600 15321 13524 873 3602 1369 11422
    7039 17579 16732 1166 9497 14498 11817 691 12683 11133 10036 10168
    9240 7540 16302 2752 10828 18058 9911 15729 17807 1719 19117 1924
    3905 6227 4868 10358 11763 18113 5209 8329 10740 6938 9635 14512
    17399 18121 12733 13966 5279 1953 1959 15567 18234 3818 5220 2744
    18925 13352 15418 7193 635 9142 8342 1452 16526 8583 14626 10595
    15517 3675 15449 18995 1550 12913 1574 10748 15207 689 10793 16997
    12087 3593 9495 12369 16764 13313 16678 1034 9702 12049 15046 15685
    1505 4817 6591 10004 17280 1057 2209 16242 6770 11621 11066 19348
    13114 5818 18606 11096 2735 11091 16599 13119 10928 11061 18170 18353
    3156 18093 8497 15157 14638 11736 1201 11263 18000 17999 11949 914
    4681 8852 14381 624 7004 5368 718 17732 11589 12259 1569 3028
    5910 8948 4159 8737 11933 15646 4723 2738 4789 376 8578 16888
    18857 6948 802 19507 5226 6977 6646 17557 2557
    374: 16075 17346 1488 14198 4254 1650 2972 17330 12199 963 19510 17391
    5693 10970 15087 18216 17575 18987 17965 11155 16523 17488 9549 11549
    10008 17518 8820 12423 1502 16789 5728 716 10447 10464 16976 5052
    6412 5108 3314 6292 8733 16269 15372 10825 12586 10413 10380 12879
    10280 19593 3339 9629 3902 1154 10253 8577 7055 2366 3283 13877
    9422 4904 10226 1456 18827 6632 11525 14700 16759 2841 3514 3755
    17006 17415 7347 3067 13384 3558 2974 1846 3323 6629 3184 19561
    19703 15961 8827 2835 9303 1071 10179 14834 435 12500 4768 8437
    7517 2760 14250 12828 10786 1268 13055 13052 12497 16738 9520 19359
    10335 2636 2886 9647 6341 6642 7315 12387 19717 11540 13887 5657
    16407 18284 16813 16160 19921 18470 14030 12523 13879 8162 18330 4380
    12845 8696 15369 16716 14346 16690 4719 3277 5678 5680 10403 3623
    5774 2508 1744 19808 4467 13035 11579 7401 2215 4902 11210 11968
    15518 8984 3055 3245 18372 7421 8387 11179 7162 12630 5371 6163
    5316 14827 17771 590 15838 18775 7975 11865 15784 2067 15507 17191
    10843 14530 15228 9977 13811 16873 2501 8524 6493 9867 12072 8122
    14458 2666 15333 9399 4828 15607 12871 15613 1771 16671 7034 11542
    15229 10987 6960 14341 14525 7247 4763 19509 5283 18405 8785 16222
    14297 18434 14175 18636 10459 13065 17257 16561 1889 14928 6483 19913
    2456 1117 3838 6107 6860 7721 6402 1845 12187 10183 3985 7714
    6914 17425 9651 13259 791 15634 15360 10630 8039 9174 19497 1774
    12575 11290 11928 14723 17308 16624 18115 9049 7707 4914 15036 6484
    13266 7824 18137 7109 4623 13337 19831 7899 18876 14992 15343 4660
    14819 3465 1564 18556 19844 4349 19085 9971 13851 16931 2174 8283
    19354 11651 4757 15968 6959 10967 11188 17645 8006 6247 8959 10307
    13566 11039 13744 17853 863 11617 7364 10342 548 17035 13825 1142
    13781 9657 5941 1581 17069 12968 15470 16299 4281 15614 1937 4559
    14610 4696 13538 7697 3637 7603 11413 2179 7933 19467 8355 1987
    19608 967 15718 3480 8769 12950 16296 15497 18747 16455 2726 19646
    1232 9750 12105 12718 12852 5921 12836 3388 7411 16468 8997 11368
    1122 7595 14296 9278 19558 12461 14633 16726 17802 7427 18821 8484
    8671 7208 18639 13316 13956 4535 18240 4560 14766 13755 10837 5042
    14741 10899 6606 836 5977 3221 7913 9252 12441 5332 6266 4788
    11839 7268 17954 2356 3394 7563 11666 4588 13840 956 15465 19389
    17489 4423 4266 13131 10548
    375: 16510 13467 14806 18916 13489 19287 6963 11609 12123 6168 14647 17406
    19726 10559 16928 16901 7555 12727 15736 13018 2908 998 11571 14927
    11630 8370 8372 13218 10996 17016 7958 9384 13045 13447 8144 1147
    15001 2276 2823 913 14592 3364 18232 14988 977 13106 11518 14539
    12112 17129 2915 10863 1580 4515 5798 8253 8082 416 19385 19171
    1100 16820 6199 4571 18783 1742 10182 13173 16635 17155 13139 6972
    5995 17971 7440 13865 9190 7070 17784 2010 723 15385 11201 10394
    4498 13493 13320 14352 678 13273 448 5709 11312 18597 11116 7510
    15024 12386 4804 16278 7734 1363 7445 19533 12959 1292 16198 16824
    12162 2158 10445 8558 12176 1883 5802 17888 7202 9357 9212 9289
    15706 15848 18962 11998 16594 7471 9904 6557 5534 7297 14395 12716
    14630 1709 8057 5106 13882 13240 3414 8391 13833 18908 11344 5879
    12334 16882 8131 13298 15056 11047 2998 9898 12476 14335 16430 17095
    2214 1606 8257 16665 13613 6921 7748 14323 15979 5733 12116 6947
    8474 13708 15492 18185 6299 13610 18148 14215 18714 2930 12129 15295
    15268 14650 18384 18143 3511 6197 6361 17944 17442 5229 2611 565
    16358 16357 15084 16089 1181 17594 9098 2241 19299 12023 7283 10377
    6819 4949 8736 13205 2694 19763 13769 19621 13407 9290 16521 1825
    10029 12822 2903 15953 15986 11911 13793 11546 2234 14881 9908 11523
    15533 17577 11068 17517 17721 17374 10696 12227 19181 13058 8185 15758
    4153 877 8465 19011 4495 6613 16284 8138 18523 4389 15763 3113
    17826 2647 1098 10483 10463 19364 11397 11401 11381 6868 15127 1474
    16902 5599 19363 17325 9309 19361 5037 4928 3787 16923 4146 16939
    19535 15773 19339 15539 15125 18484 4203 18687 9493 10628 16954 18751
    5417 19261 19262 19234 1714 19898 19336 19855 19291 19286 19880 19877
    19295 19895 19875 1295 10357 6086 10734 11846 6683 12722 18816 18796
    7791 4566 18951 15711 11715 3398 18259 16246 1255 3813 1431 15683
    7433 3767 5490 5324 10201 9680 13775 10924 11448 5618 3801 6703
    2837 11877 2626 19090 4080 8371 9573 3492 13188 3045 16273 5297
    2403 2365 6847 7359 12126 16429 13888
    376: 11821 15570 15362 17684 12417 16687 6089 1359 9786 19736 8877 10254
    5707 15924 3173 5784 19735 9392 2552 11302 15018 18422 14812 5768
    9824 19506 10715 19649 3163 9998 2058 6584 7848 14615 15185 2020
    15445 16636 11375 18820 10331 1794 6619 8395 15426 6787 6658 7293
    10195 2815 19672 5744 14420 15534 15370 14303 15177 12323 14958 14797
    11491 452 5665 6655 9310 16947 3284 15338 17037 6312 3537 1320
    3839 8693 1070 13922 11570 9105 5536 5454 15484 14763 14487 9068
    12682 9682 10905 17502 4201 4406 8085 8086 7056 8194 4289 2788
    19086 10609 17621 1276 11826 13369 15331 5251 8761 8829 15454 5769
    6808 15129 1725 10525 13488 7737 8203 2464 17537 3566 8056 4191
    16861 7136 8740 1328 6582 7383 9441 7191 15884 17130 5403 5900
    19080 10011 3547 16174 11235 17910 5888 18291 17740 7117 12383 15552
    8043 8613 9367 10541 17801 13465 8616 10220 1388 8599 4600 15321
    13524 873 3602 1369 11422 7039 16732 1166 9497 14498 11817 691
    12683 11133 10036 10168 9240 7540 16302 2752 10828 18058 9911 15729
    17807 1719 19117 1924 3905 6227 4868 10358 11763 18113 5209 8329
    10740 6938 14512 17399 18121 12733 13966 5279 1953 15567 3818 2744
    18925 13352 15418 7193 635 9142 8342 1452 16526 8583 14626 10595
    15517 3675 15449 18995 1550 12913 1574 10748 15207 689 10793 12087
    9495 12369 16764 13313 16678 1034 9702 12049 15046 9046 15685 1505
    4817 6591 10004 17280 18300 1057 2209 13541 16242 6770 17146 11621
    11066 2735 11091 11096 19348 10928 13114 5818 18353 3156 18093 15157
    11263 11949 914 4681 7472 8852 624 7004 5368 718 17732 12259
    3028 1569 5910 8948 4159 5738 8737 15646 4723 4789 12067 16888
    18857 373 6948 802 5226 6977 6646 17557 2557
    377: 8393 9015 13413 2587 10796 17631 11121 634 433 4963 9556 575
    5319 15391 10530 431 9031 4635 9004 9073 3306 11645 3981 19141
    19101 19812 10554 16183 13822 14492 17246 1376 15351 19391 12231 4365
    5194 2111 5408 17546 18797 3789 5903 1218 4848 19643 6748 4899
    1341 2965 13343 10436 3965 12507 16052 10080 14622 1156 15431 5027
    7279 4985 13179 3070 3071 18612 2494 9230 6746 8566 5265 5208
    3815 11698 12148 18164 13048 16879 8300 4441 9892 11252 16442 505
    453 12406 1723 6094 17550 19037 5884 16347 17111 14923 4421 18481
    6530 12498 17991 3134 8890 3350 7985 7864 10001 10340 991 5289
    19317 10465 7319 2659 5841 13312 1585 8483 2167 16410 18310 17967
    11530 8907 847 867 8377 12935 12438 1630 8528 11657 18567 17817
    16778 524 942 2110 13396 13561 12813 15308 14883 14448 5321 8706
    4558 4612 4061 16274 14724 2777 607 3112 6814 11242 4393 19491
    1312 1238 437 18920 2620 5133 9619 8260 13014 8506 10965 3168
    2373 14342 18448 19165 17471 5405 6607 1741 14959 15075 16265 13994
    1258 14304 8744 10343 19837 12926 7213 3174 10993 14602 10617 7779
    10187 9485 15515 19398 18257 19349 1202 15472 7901 12094 13651 1646
    14731 1180 12154 16047 18288 7639 2248 8051 7996 7995 3855 1551
    14194 18409 5592 11704 8432 13409 18199 12052 7391 8999 15302 4964
    15580 16350 15735 14708 15312 6145 15699 6159 15221 18322 10493 501
    6886 13963 12295 8982 13436 16066 11980 4664 9057 9034 16666 15687
    2427 11094 13668 3320 14079 9090 11181 8003 4257 11638 6544 6957
    3999 12296 10381 2653 4772 4810 8751 10568 10162 5600 5649 15893
    14376 16307 17697 9785 10884 16276 6169 6176 1037 9196 8367 7783
    14533 9056 15139 19917 12676 3038 10673 5503 7023 15682 10198 898
    1297 5858 14777 6955 17092 945 3032 16741 4106 11060 10054 18270
    12505 13314 1821 7784 11537 6572 3603 13635 5021 9052 7601 6284
    8815 10431 18883 18773 17616 11724 3501 16356 11013 14805 14557 7384
    17109 7703 14347 18849 5364 8936 3979 19281 10693 13957 3977 7380
    13486 3384
    378: 13086 19930 5574 17164 15382 2809 1565 9389 817 831 5123 14538
    9536 1834 1833 13082 18812 19542 12765 13660 1610 1609 15422 15421
    1627 13773 5445 16272 10838 19127 19142 9628 10532 7251 8084 9179
    10060 3233 3234 10382 15456 12062 12060 8373 598 11569 18041 16210
    3876 5308 5310 4105 19053 17469 5866 18520 18518 17299 16592 13763
    1649 2826 1088 5244 17633 8002 6325 8510 8509 17342 10265 8523
    11182 2722 2708 14181 17828 10012 8552 6241 10590 5676 10274 8661
    3295 6576 8917 12079 16538 11322 11319 15823 16815 16795 13353 14432
    2003 1985 7807 7667 1870 12245 15011 15010 7081 7327 7326 3951
    11419 11420 5668 5402 2091 2088 10840 16194 9002 9003 8127 8141
    1440 6106 6104 17126 17005 4082 9591 9590 1915 6725 10684 7300
    3317 3316 9330 19487 19488 14152 14153 2122 1914 1911 10665 14295
    14309 2813 2489 17345 11755 18173 14628 16281 4570 426 12610 17201
    17057 16060 16058 9609 987 9610 15673 921 1086 18306 11336 2498
    17087 17085 12034 12035 14678 14675 1138 544 16271 12318 12812 4308
    19378 14598 14599 7369 18594 7648 17481 19008 14345 5166 7493 13726
    13725 8384 3400 16200 7902 8410 8409 13137 16958 16957 15723 12257
    12253 15978 12973 4460 4459 13017 1570 1078 2531 15107 15091 8965
    13199 13198 19203 19218 2645 523 5806 17244 17241 9229 13301 12697
    16182 14543 17648 7956 541 7604 12160 14921 4194 19094 17637 18276
    18272 14246 14245 6265 11350 6263 11351 1447 9984 5779 16703 16701
    8272 8270 15033 637 11702 3382 13215 6987 17734 17485 17484 8448
    11578 16065 14275 12748 19807 2279 9116 2296 2779 13325 13222 5611
    2538 12956 15346 10824 1158 4300 18255 6983 15168 4680 19799 12284
    6950 16092 3445 2799 10239 3660 12422 2797 18788 11987 9273 10359
    2829 13626 19734 592 14588 3599 6112 6093 6110 572 5844 11779
    4108 3093 11517 11539 455 10947 11127 13214 3927 11199 12891 15461
    15460 7465 5719 5718 3367 13458 13456 1149 14244 6969 12138 9126
    1661 15396 5031 16825 1467 3689 6716 6528 6526 8496 18877
    379: 16324 10027 16399 19626 19600 476 10737 6503 2637 5207 7908 18645
    13757 8204 17032 1213 10125 9070 5372 15085 10478 7338 18023 9641
    11812 14642 18106 9561 2651 1666 5437 14594 5439 12210 6002 12290
    5485 6003 14353 14349 14351 10819 8846 9429 2453 10112 16543 3140
    1146 1124 1121 15438 2354
    380: 15042 683 16630 18404 5663 2482 10670 11782 18811 18709 11700 19144
    6755 9799 13818 6126 2455 8789
    381: 17923 18890 5500 4771 6745 5378 3236 1210 11603 19433 19342 15300
    17953 11454 9261 12841 11591 5415 5453 5430 8278 14564 6761 17927
    18569 14781 10539 6214 3620 3528 17899 8748 12221 5504 5502 9489
    6425 8958
    382: 19132 10614 8139 10836 16040 11545 7879 2100 1703 14547
    383: 1189 16611 3586 4641 19633 17705 17150 8849 3552 7294 2029 6153
    18596
    384: 741 1633 5923 8765 16673 14609 3698 6449 2951 2093 11563 614
    14593 14762 6397 7731 14290 17038 4311 14640
    385: 11357 13113 2101 9359 2343 5986 9703 12857 8854 17190 4889 13808
    12572 3386 7845 17983 4794
    386: 1016 16046 10106 6272 16847 10542 15062 5576 4893 5448 15407 7831
    16578 18278 2702 7215 9961 6250 3252 18599 16433 3226 17007
    387: 7223 8111 7867 1322 9479 3291 18674 5132 12538 3515 18852 16518
    14682 5419 9781
    388: 13217 19387 1319 13219 1333 2384 4322 7600 13280 5924 13688 2181
    16877 16122 5091 927 6100 3686
    389: 11269 4678 13073 1755 1754 8654 3662 17371 3891 3973 2187 15246
    18462 4802 3390 619 643 13183 4014 1542 10000 19057 6229 1262
    14305 14256 17590 5708 4877 3775 8462
    390: 17182 3673 6158 5731 8195 15335 7264 7267 5346 10728 11675 2337
    19110 7961 8491 12046 10224 6729 18918 18228 3153 8723 3309 3313
    6222 15956 4632 12325 6835 6387 2622 4496 9897 10860 8044 10003
    13262 13236 6403 5331 4640 1303 4602 1304 18435 16053 18480 17646
    17644 17675 16363 4781 3269 8610 10852 3582 5717 9242 9340 9346
    13271 1345 3780 11086 14006 2264 5062 5146 16786 891 1282 11639
    2472 9621 19732 15665 10462 16384 18138 9510 19888 19119 4931 18991
    13945 18937 13580 18401 13863 1936 4639 10245 760 13679 14995 17389
    5259 10385 9763 15381 10901 10886 10888 12415 15437 8840 7871 4684
    12209 17107 11203 16435 8624 13410 8530 10422 11650 15110 13639 12675
    13689 19825 4662 13277 5625 9279 3369 5976 1377 2144 4086 18044
    16889 6767 18418 11859 8188 6500 16529 16528 13234 14259 18077 11012
    10071 11825 16181 6479 3800 9154 19377 924 923 7361 4574 14380
    13895 17168 14901 17969 13735 17599 10270 4099 18973 3523 14337 8378
    4490 18286 5203 2114 13850 9109 9096 7060 4161 6561 1211 9640
    13790 19180 14796 5771 15355 14027 6282 12681 16502 4842 4262 4930
    15500 16490 14450 18361 15929 1171 7624 13836 15083 19541 1490 8022
    9426 19755 13700 10435 8346 13719 8681 19368 10906 19116 1509 3865
    12853 3125 5889 8316 18315 9077 436 1977 8354 5002 5025 16553
    5171 17152 9352 17908 9960 9963 4907 2079 14164 3744 3102 18375
    4128 4129 14794
    391: 5830 10547 4780 6670 1560 15309 12977 11581 15243 10500 7299 19550
    4614 11584 18988 17503 13880 18201 7124 6036 17359 15414 19074 13696
    4180 810 12806 13939 1259 2434 12029 5666 8718 751 18491 15717
    1795 4475 1738 627 5732 18253 8660 11107 16309 12868 4299 14698
    14679 14502 8248 11990 9758 16167 9385 10043 10430 16906 12099 5791
    16810 13759 8562 2921 8889 10315 14077 2682 18457 14406 12241 10194
    5832 13548 15304 4676
    392: 18452 9432 5303 14233 9970 7637 14980 15265 7581 3016 10412 19809
    393: 19384 19382 682 6463 1021 6039 6041 6057 7898 7896 13532 13533
    8037 9356 16887 16866 7150 7148 11880 11903 7084 7083 3381 13816
    13817 12463 4530 2311 2312 10624 10623 15054 2256 2257 14016 14018
    8801 6140 6142 8800 6138 8357 8375 8360 3666 3668 11044 11022
    9473 9494 5001 5000 4831 4836 18327 19232 5812 5815 9999 7941
    7942 749 3079 7426 7451 8001 7999 8005 19635 4051 15885 12205
    2860 786 784 15576 15575 12310 12307 2306 3461 7640 10242
    394: 12840 13577 8132 1535 5905 3211 3797 12558 6843 17949 15017 9231
    1310 17041 6367 18764 8461 15888 6866 6864 7983 8980 10079 15489
    11102 9933 7325 1749 8441 7720 17727
    • Example 3 Consensus Sequence Build
  • ClustalW program is selected for multiple sequence alignments of an amino acid sequence of SEQ ID NO: 198 and its homologs, through SEQ ID NO: 394 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: 227 and its 25 homologs were derived according to the procedure described above and is displayed in FIG. 1.
    • Example 4 Identification of Amino Acid Domain by Pfam Analysis
  • This example illustrates the identification of amino acid domain 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 protein families for the proteins of SEQ ID NO: 198 through 394 are shown in Table 16. 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: 207 is characterized by two Pfam domains, i.e. “TFIIS_M” and “TFIIS_C”. See also the protein with amino acids of SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”. In Table 16 “score” is the gathering score for the Hidden Markov Model of the domain which exceeds the gathering cutoff reported in Table 17.
  • TABLE 16
    PEP SEQ Pfam domain
    ID NO GENE ID name begin stop score E-value
    198 CGPG106 CMAS 69 306 −149.8 5.00E−05
    198 CGPG106 Methyltransf_11 131 229 109.1 1.20E−29
    198 CGPG106 Methyltransf_12 131 227 54.6 3.10E−13
    200 CGPG117 SNF5 14 252 66.9 5.90E−17
    202 CGPG1288 Cupin_3 57 131 130.8 3.40E−36
    204 CGPG1458 Phosphorylase 180 958 1454.7 0
    205 CGPG1542 LMBR1 11 488 715.1 4.40E−212
    206 CGPG170 Metallophos 54 249 140.4 4.50E−39
    207 CGPG1828 TFIIS_M 206 327 203.5 4.50E−58
    207 CGPG1828 TFIIS_C 338 376 83.3 7.00E−22
    209 CGPG2217 ORMDL 14 156 356.8 3.20E−104
    210 CGPG2292 Tim17 52 186 16.1 4.20E−05
    210 CGPG2292 SAM_1 192 253 13.8 0.019
    212 CGPG2499 Glycolytic 4 339 588.8 4.50E−174
    213 CGPG2653 WD40 135 172 40.3 6.20E−09
    213 CGPG2653 WD40 235 271 40.7 4.60E−09
    213 CGPG2653 WD40 276 313 38 3.00E−08
    214 CGPG2813 HLH 204 251 34.1 4.60E−07
    215 CGPG3002 eIF-4B 5 451 −50.3 1.90E−10
    216 CGPG3154 C2 14 102 101.8 1.80E−27
    216 CGPG3154 C2 198 272 57.8 3.20E−14
    217 CGPG3235 B_lectin 66 176 156.8 5.30E−44
    217 CGPG3235 S_locus_glycop 189 315 227 3.90E−65
    217 CGPG3235 PAN_2 332 402 103.9 4.20E−28
    217 CGPG3235 Pkinase_Tyr 488 757 75.6 2.10E−20
    217 CGPG3235 Pkinase 488 757 109 1.30E−29
    218 CGPG3274 Allene_ox_cyc 74 253 401.6 1.10E−117
    219 CGPG3275 Auxin_inducible 50 155 135.6 1.30E−37
    220 CGPG3363 zf-AN1 21 58 10.5 0.0033
    220 CGPG3363 zf-AN1 103 148 12.4 0.0019
    221 CGPG3367 TFIIA 9 375 556 3.40E−164
    222 CGPG3375 Tryp_alpha_amyl 33 111 55.2 1.90E−13
    224 CGPG3534 Ank 42 74 13.6 0.31
    224 CGPG3534 Ank 75 107 36.8 6.90E−08
    224 CGPG3534 Ank 108 140 1.6 17
    224 CGPG3534 Pkinase_Tyr 160 413 202.4 9.90E−58
    224 CGPG3534 Pkinase 160 413 198.5 1.40E−56
    225 CGPG3638 Ribonuclease_T2 30 219 367.3 2.20E−107
    226 CGPG3918 Metallophos 60 255 159.1 1.10E−44
    229 CGPG3972 14-3-3 8 243 501.1 1.20E−147
    230 CGPG3990 zf-C3HC4 259 299 47.3 4.60E−11
    231 CGPG3994 Pkinase 554 802 207 4.10E−59
    231 CGPG3994 Pkinase_Tyr 554 802 233 5.90E−67
    232 CGPG4026 CMAS 59 320 −174.5 0.003
    232 CGPG4026 Methyltransf_11 122 220 96.3 8.10E−26
    232 CGPG4026 Methyltransf_12 122 218 37 5.90E−08
    232 CGPG4026 Sterol_MT_C 229 358 301.1 1.80E−87
    233 CGPG4048 Pkinase 21 275 342.1 8.80E−100
    233 CGPG4048 Pkinase_Tyr 21 275 65.1 1.20E−19
    233 CGPG4048 NAF 304 364 104.5 2.80E−28
    234 CGPG4052 DUF298 127 242 222.4 9.20E−64
    236 CGPG4058 Asp 99 437 −128.4 4.60E−06
    237 CGPG4069 adh_short 31 218 −16.7 4.80E−05
    239 CGPG4088 SWIB 324 399 96.4 7.80E−26
    241 CGPG4121 Cyclin_N 58 190 133.7 4.80E−37
    241 CGPG4121 Cyclin_C 192 314 44.3 3.70E−10
    242 CGPG4122 zf-C3HC4 100 141 27.6 4.10E−05
    243 CGPG4140 p450 48 484 140.8 3.30E−39
    244 CGPG4154 zf-CCCH 90 115 22.6 0.0012
    245 CGPG4311 FBPase 70 395 347.9 1.50E−101
    247 CGPG4369 BoIA 10 79 92.1 1.60E−24
    248 CGPG442 2-Hacid_dh 85 394 140.9 3.20E−39
    248 CGPG442 2-Hacid_dh_C 187 362 293 5.20E−85
    248 CGPG442 ACT 551 621 39.9 8.10E−09
    249 CGPG4454 p450 45 447 −35.5 2.10E−09
    250 CGPG4456 p450 75 502 103.6 5.30E−28
    252 CGPG4588 Auxin_inducible 1 102 151.5 2.00E−42
    253 CGPG4765 DUF868 28 304 175.5 1.20E−49
    255 CGPG4912 WD40 243 281 31 3.80E−06
    256 CGPG4926 WD40 42 79 22.8 0.0012
    256 CGPG4926 WD40 126 163 25.8 0.00014
    257 CGPG4967 Asp 71 424 −94.7 4.70E−08
    258 CGPG4977 Usp 3 157 85.3 1.80E−22
    259 CGPG5001 adh_short 5 181 6.4 1.40E−06
    260 CGPG5025 adh_short 30 212 5.7 1.50E−06
    261 CGPG5041 DUF26 77 132 84.3 3.40E−22
    261 CGPG5041 DUF26 188 242 100.7 4.00E−27
    261 CGPG5041 Pkinase 333 558 60.3 5.70E−15
    262 CGPG5116 ArfGap 15 137 174.1 3.20E−49
    262 CGPG5116 C2 182 261 101.4 2.40E−27
    263 CGPG5144 p450 61 535 157.9 2.40E−44
    264 CGPG5171 p450 35 496 137 4.60E−38
    265 CGPG5194 DUF1191 25 308 616.2 2.60E−182
    268 CGPG5221 p450 30 499 135.4 1.40E−37
    269 CGPG5269 PCI 297 401 108.9 1.30E−29
    270 CGPG5404 Peptidase_S10 68 480 683.3 1.70E−202
    271 CGPG5432 MtN3_slv 12 99 145.4 1.40E−40
    271 CGPG5432 MtN3_slv 133 219 140.4 4.40E−39
    272 CGPG5518 Ribosomal_S8e 1 238 325.9 6.30E−95
    273 CGPG5535 WD40 335 371 28.8 1.80E−05
    273 CGPG5535 WD40 434 471 37.1 5.50E−08
    273 CGPG5535 WD40 476 513 47.2 5.20E−11
    273 CGPG5535 WD40 518 555 35.6 1.60E−07
    273 CGPG5535 WD40 567 604 36.1 1.10E−07
    273 CGPG5535 WD40 621 658 34.9 2.50E−07
    273 CGPG5535 WD40 663 706 35.5 1.60E−07
    274 CGPG5540 ESCRT-III 21 207 265.7 8.40E−77
    275 CGPG5568 AA_permease 69 521 −67.1 0.00013
    276 CGPG5577 SMC_N 21 1049 −2.8 1.50E−11
    277 CGPG5587 Thioredoxin 78 199 0.1 0.00015
    278 CGPG5594 Histone 27 100 99.6 8.80E−27
    279 CGPG5633 PGAM 91 277 153.2 6.30E−43
    280 CGPG5640 Aminotran_1_2 30 385 362.1 8.00E−106
    281 CGPG5646 iPGM_N 2 363 876.8 9.10E−261
    281 CGPG5646 Metalloenzyme 373 488 173.3 5.40E−49
    282 CGPG5656 Gln-synt_N 17 99 85.7 1.30E−22
    282 CGPG5656 Gln-synt_C 105 358 528.1 8.50E−156
    283 CGPG5659 Aminotran_3 35 361 321.7 1.20E−93
    284 CGPG5661 PK 1 345 702 3.80E−208
    284 CGPG5661 PK_C 355 469 115.4 1.50E−31
    285 CGPG5684 Glycolytic 4 339 578.6 5.60E−171
    286 CGPG5694 TIM 4 246 465.9 4.50E−137
    287 CGPG5704 NDK 2 136 329 7.30E−96
    288 CGPG5714 NDK 4 138 266.4 5.20E−77
    289 CGPG5721 Rib_5-P_isom_A 47 215 354.4 1.70E−103
    290 CGPG5728 zf-CCCH 80 106 39.3 1.20E−08
    291 CGPG5757 Sad1_UNC 203 330 195.6 1.00E−55
    292 CGPG5764 Actin 11 140 36.4 6.60E−08
    293 CGPG5783 TPT 272 442 71.5 2.40E−18
    294 CGPG5791 AA_permease 114 588 704.9 5.40E−209
    295 CGPG5799 Aa_trans 206 601 453 3.60E−133
    296 CGPG5856 Pkinase 79 353 159.5 8.00E−45
    296 CGPG5856 Pkinase_Tyr 79 353 149.4 9.00E−42
    297 CGPG5927 AAA 58 248 252.4 8.70E−73
    297 CGPG5927 AAA 322 510 290.8 2.40E−84
    298 CGPG5941 PfkB 42 336 86.2 9.00E−23
    299 CGPG5957 CBM_20 86 178 23 5.70E−07
    300 CGPG5967 DUF822 2 147 307.1 2.90E−89
    301 CGPG6040 LEA_3 1 88 178.7 1.30E−50
    302 CGPG607 PurA 28 275 44.4 5.80E−12
    303 CGPG6178 DUF1336 53 267 427.9 1.30E−125
    304 CGPG6185 UQ_con 7 148 197.9 2.10E−56
    305 CGPG6306 APC8 1 161 401.5 1.10E−117
    305 CGPG6306 TPR_1 339 372 34.7 2.90E−07
    305 CGPG6306 TPR_2 339 372 23.8 0.00058
    305 CGPG6306 TPR_2 373 406 23.7 0.00062
    305 CGPG6306 TPR_1 373 406 34.1 4.40E−07
    305 CGPG6306 TPR_2 407 440 22 0.0019
    305 CGPG6306 TPR_1 407 440 24.3 0.00039
    306 CGPG6318 MFS_1 31 496 35.3 1.90E−07
    306 CGPG6318 PTR2 92 484 208.6 1.30E−59
    307 CGPG6326 Kelch_1 34 79 45 2.40E−10
    307 CGPG6326 Kelch_2 34 79 43.5 6.80E−10
    307 CGPG6326 Kelch_1 152 198 26.7 7.70E−05
    307 CGPG6326 Kelch_2 152 198 32.1 1.80E−06
    307 CGPG6326 Kelch_2 203 249 20.2 0.0067
    307 CGPG6326 Kelch_1 204 248 7.9 0.45
    308 CGPG6370 Gp_dh_N 3 151 326 5.80E−95
    308 CGPG6370 Gp_dh_C 156 313 362 8.80E−106
    309 CGPG6429 ADH_N 25 135 159.4 8.40E−45
    309 CGPG6429 ADH_zinc_N 166 305 105.1 1.90E−28
    310 CGPG6440 PK 1 345 789.4 1.90E−234
    310 CGPG6440 PK_C 357 471 167.5 3.10E−47
    310 CGPG6440 PEP-utilizers 486 575 134 3.60E−37
    311 CGPG6516 Aldedh 19 478 778.3 4.10E−231
    312 CGPG6653 LRRNT_2 23 66 49.3 1.20E−11
    312 CGPG6653 LRR_1 71 93 12.1 1.4
    312 CGPG6653 LRR_1 95 117 10.5 2.8
    312 CGPG6653 LRR_1 119 142 13 0.93
    312 CGPG6653 LRR_1 144 166 19.5 0.011
    312 CGPG6653 LRR_1 168 190 10.6 2.7
    312 CGPG6653 LRR_1 192 214 8.8 5.7
    312 CGPG6653 LRR_1 289 311 17.4 0.046
    312 CGPG6653 LRR_1 313 335 10.8 2.4
    312 CGPG6653 LRR_1 337 359 10.7 2.5
    312 CGPG6653 LRR_1 361 384 12 1.5
    312 CGPG6653 LRR_1 409 431 10.4 2.9
    312 CGPG6653 LRR_1 457 479 11.9 1.5
    312 CGPG6653 LRR_1 481 503 10.4 3
    312 CGPG6653 LRR_1 505 527 10.5 2.8
    312 CGPG6653 LRR_1 529 551 11.1 2.2
    312 CGPG6653 LRR_1 553 575 9.3 4.7
    312 CGPG6653 LRR_1 577 598 11.1 2.1
    312 CGPG6653 Pkinase 695 966 134.8 2.20E−37
    312 CGPG6653 Pkinase_Tyr 695 966 134.5 2.60E−37
    313 CGPG6712 PGAM 91 277 154.6 2.30E−43
    314 CGPG6737 PGAM 92 253 173 6.80E−49
    315 CGPG6747 FBPase 106 429 448.6 7.20E−132
    316 CGPG6796 Alpha-amylase 14 452 199.5 7.00E−57
    318 CGPG6810 GH3 12 561 1261.5 0
    320 CGPG6953 Ank 121 153 50.4 5.60E−12
    321 CGPG7121 L51_S25_CI-B8 20 93 108.9 1.40E−29
    322 CGPG7163 Prenylcys_lyase 149 500 788 5.20E−234
    324 CGPG7206 Aldo_ket_red 14 298 389.4 4.80E−114
    325 CGPG7225 Subtilisin_N 48 125 84.3 3.30E−22
    326 CGPG7267 DUF588 34 164 154.6 2.40E−43
    327 CGPG7272 DUF1005 50 254 524.8 8.50E−155
    328 CGPG7281 FA_hydroxylase 86 229 361.9 9.30E−106
    329 CGPG7308 CoA_binding 1 100 −10.8 0.04
    329 CGPG7308 NAD_Gly3P_dh_N 4 147 −0.6 2.00E−06
    329 CGPG7308 F420_oxidored 5 251 282.5 7.50E−82
    330 CGPG7316 Anti-silence 1 155 419.9 3.20E−123
    331 CGPG7371 Response_reg 29 157 94.8 2.30E−25
    332 CGPG7457 PfkB 114 408 146.6 6.00E−41
    335 CGPG7636 LSM 13 81 78.2 2.40E−20
    338 CGPG7804 FAR1 62 279 364.9 1.20E−106
    338 CGPG7804 SWIM 556 589 37.3 4.90E−08
    339 CGPG7823 Rotamase 104 188 91.8 1.90E−24
    339 CGPG7823 Rhodanese 203 298 44.1 4.40E−10
    340 CGPG7828 DnaJ 12 81 66.1 1.00E−16
    340 CGPG7828 zf-CSL 96 174 25.2 0.00021
    343 CGPG7986 F-box 48 96 31.2 3.40E−06
    343 CGPG7986 LRR_1 189 216 9 5.4
    343 CGPG7986 LRR_1 428 451 8.2 7.7
    343 CGPG7986 LRR_1 561 584 8.4 6.9
    345 CGPG8015 zf-CCHC 18 35 24.2 7.90E−05
    349 CGPG8083 Tryp_alpha_amyl 28 105 37.6 3.90E−08
    350 CGPG8106 BURP 56 280 380 3.20E−111
    353 CGPG8152 PAP_fibrillin 9 124 45.9 1.20E−10
    354 CGPG8166 PBD 118 166 49.9 7.80E−12
    355 CGPG8377 Oleosin 30 109 55 2.20E−13
    356 CGPG8976 Ceramidase_alk 50 795 1545.5 0
    357 CGPG8987 FH2 439 839 552.9 3.10E−163
    358 CGPG9013 NAD_binding_1 234 350 138.6 1.50E−38
    359 CGPG9080 EGF_CA 315 357 42.2 1.60E−09
    359 CGPG9080 Pkinase 433 716 120.3 4.90E−33
    359 CGPG9080 Pkinase_Tyr 433 704 113.1 7.40E−31
    360 CGPG9081 DUF676 30 247 319 7.50E−93
    361 CGPG9130 MMR_HSR1 266 369 71.9 1.80E−18
    362 CGPG9133 PPR 125 159 13.2 0.19
    362 CGPG9133 PPR 161 195 2.8 3.2
    362 CGPG9133 PPR 196 230 22.7 0.0012
    362 CGPG9133 PPR 232 266 42.1 1.70E−09
    362 CGPG9133 PPR 267 301 29.4 1.20E−05
    362 CGPG9133 PPR 302 336 49.4 1.10E−11
    362 CGPG9133 PPR 337 371 32.9 1.00E−06
    362 CGPG9133 PPR 372 407 7.9 0.8
    362 CGPG9133 PPR 408 442 49.7 8.80E−12
    362 CGPG9133 PPR 443 477 22.6 0.0013
    362 CGPG9133 PPR 478 512 37.8 3.40E−08
    362 CGPG9133 PPR 513 547 42.9 1.00E−09
    362 CGPG9133 PPR 548 582 30.2 6.70E−06
    362 CGPG9133 PPR 583 617 38.4 2.20E−08
    363 CGPG9134 HD 91 232 46.2 1.00E−10
    364 CGPG9137 RrnaAD 65 337 122.2 1.30E−33
    365 CGPG9141 Pantoate_transf 40 306 402.9 4.40E−118
    366 CGPG9145 Lung_7-TM_R 168 423 385.2 8.80E−113
    367 CGPG9147 DNA_pol_E_B 178 389 249.9 5.00E−72
    368 CGPG9148 p450 36 502 128.8 1.40E−35
    369 CGPG9155 Pkinase 86 347 56.9 6.10E−14
    369 CGPG9155 Pkinase_Tyr 86 351 73.7 2.90E−20
    370 CGPG9163 Na_H_Exchanger 12 378 280.1 3.80E−81
    370 CGPG9163 TrkA_N 416 531 118.8 1.50E−32
    371 CGPG9170 Complex1_30 kDa 90 158 103.8 4.70E−28
    371 CGPG9170 Complex1_49 kDa 298 537 2.8 6.10E−13
    373 CGPG9183 HTH_11 1 56 72.4 1.30E−18
    373 CGPG9183 BPL_LipA_LipB 84 182 94.5 2.90E−25
    373 CGPG9183 BPL_C 275 322 43.3 7.80E−10
    374 CGPG9186 DHBP_synthase 8 203 370.6 2.20E−108
    374 CGPG9186 GTP_cyclohydro2 208 366 −2.3 3.80E−10
    375 CGPG9205 NTP_transferase 4 288 421 1.50E−123
    375 CGPG9205 MannoseP_isomer 299 465 350.8 2.00E−102
    375 CGPG9205 Cupin_2 380 450 55.3 1.80E−13
    376 CGPG9207 HTH_11 6 59 50.9 3.90E−12
    376 CGPG9207 BPL_LipA_LipB 83 180 104.5 2.80E−28
    376 CGPG9207 BPL_C 271 317 46.5 8.50E−11
    377 CGPG9219 Complex1_30 kDa 107 175 99.8 7.60E−27
    377 CGPG9219 Complex1_49 kDa 311 537 −11.6 5.90E−12
    378 CGPG9220 GDC-P 3 443 700.9 8.30E−208
    379 CGPG9230 Peptidase_S10 88 488 657.2 1.20E−194
    381 CGPG9238 Tryp_alpha_amyl 36 114 56 1.20E−13
    382 CGPG9259 Mit_rib_S27 14 93 135.3 1.50E−37
    383 CGPG9271 NPH3 215 418 189.9 5.60E−54
    384 CGPG9275 ETC_C1_NDUFA5 35 91 112.8 9.00E−31
    386 CGPG9283 DUF1195 6 161 180.3 4.30E−51
    387 CGPG9309 MAP65_ASE1 38 575 52.8 1.10E−12
    389 CGPG9322 Pkinase 103 383 170 5.40E−48
    389 CGPG9322 Pkinase_Tyr 103 383 158.7 1.30E−44
    390 CGPG9335 Sugar_tr 7 726 210.6 3.30E−60
    390 CGPG9335 MFS_1 11 685 107.7 3.00E−29
    391 CGPG9341 RMMBL 531 573 35.9 1.20E−07
    392 CGPG9344 TPR_2 531 564 23.9 0.00051
    393 CGPG9345 UPF0261 5 432 483.7 2.00E−142
    394 CGPG976 Glyco_transf_8 216 533 401.3 1.30E−117
  • TABLE 17
    Pfam domain accession gathering
    name number cutoff domain description
    14-3-3 PF00244.9 25 14-3-3 protein
    2-Hacid_dh PF00389.19 11.2 D-isomer specific 2-hydroxyacid
    dehydrogenase, catalytic domain
    2-Hacid_dh_C PF02826.7 −82.2 D-isomer specific 2-hydroxyacid
    dehydrogenase, NAD binding domain
    AAA PF00004.18 12.3 ATPase family associated with various
    cellular activities (AAA)
    AA_permease PF00324.10 −120.8 Amino acid permease
    ACT PF01842.13 0 ACT domain
    ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain
    ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase
    APC8 PF04049.3 −19.8 Anaphase promoting complex subunit 8/
    Cdc23
    Aa_trans PF01490.7 −128.4 Transmembrane amino acid transporter
    protein
    Actin PF00022.8 −30 Actin
    Aldedh PF00171.11 −209.3 Aldehde dehydrogenase family
    Aldo_ket_red PF00248.10 −97 Aldo/keto reductase family
    Allene_ox_cyc PF06351.2 25 Allene oxide cyclase
    Alpha-amylase PF00128.12 −93 Alpha amylase, catalytic domain
    Aminotran_1_2 PF00155.10 −57.5 Aminotransferase class I and II
    Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III
    Ank PF00023.18 0 Ankyrin repeat
    Anti-silence PF04729.4 25 Anti-silencing protein, ASF1-like
    ArfGap PF01412.8 −17 Putative GTPase activating protein for Arf
    Asp PF00026.13 −186.1 Eukaryotic aspartyl protease
    Auxin_inducible PF02519.4 −15 Auxin responsive protein
    BPL_C PF02237.6 16 Biotin protein ligase C terminal domain
    BPL_LipA_LipB PF03099.8 −0.2 Biotin/lipoate A/B protein ligase family
    BURP PF03181.5 −52 BURP domain
    B_lectin PF01453.14 28.2 D-mannose binding lectin
    BolA PF01722.7 23 BolA-like protein
    C2 PF00168.18 3.7 C2 domain
    CBM_20 PF00686.9 −3 Starch binding domain
    CMAS PF02353.10 −177.9 Cyclopropane-fatty-acyl-phospholipid
    synthase
    Ceramidase_alk PF04734.3 25 Neutral/alkaline non-lysosomal ceramidase
    CoA_binding PF02629.8 −12.8 CoA binding domain
    Complex1_30kDa PF00329.8 −3 Respiratory-chain NADH dehydrogenase, 30 Kd
    subunit
    Complex1_49kDa PF00346.8 −108 Respiratory-chain NADH dehydrogenase, 49 Kd
    subunit
    Cupin_2 PF07883.1 16.6 Cupin domain
    Cupin_3 PF05899.2 4.4 Protein of unknown function (DUF861)
    Cyclin_C PF02984.8 −13 Cyclin, C-terminal domain
    Cyclin_N PF00134.13 −14.7 Cyclin, N-terminal domain
    DHBP_synthase PF00926.10 −116 3,4-dihydroxy-2-butanone 4-phosphate
    synthase
    DNA_pol_E_B PF04042.5 −47.5 DNA polymerase alpha/epsilon subunit B
    DUF1005 PF06219.2 25 Protein of unknown function (DUF1005)
    DUF1191 PF06697.2 25 Protein of unknown function (DUF1191)
    DUF1195 PF06708.1 25 Protein of unknown function (DUF1195)
    DUF1336 PF07059.2 −78.2 Protein of unknown function (DUF1336)
    DUF26 PF01657.7 0 Domain of unknown function DUF26
    DUF298 PF03556.6 25 Domain of unknown function (DUF298)
    DUF588 PF04535.2 25 Domain of unknown function (DUF588)
    DUF676 PF05057.4 −60.7 Putative serine esterase (DUF676)
    DUF822 PF05687.3 25 Plant protein of unknown function (DUF822)
    DUF868 PF05910.2 25 Plant protein of unknown function (DUF868)
    DnaJ PF00226.19 −8 DnaJ domain
    EGF_CA PF07645.4 24.5 Calcium binding EGF domain
    ESCRT-III PF03357.10 −35.4 ESCRT-III complex subunit
    ETC_C1_NDUFA5 PF04716.3 25 ETC complex I subunit conserved region
    F-box PF00646.21 13.6 F-box domain
    F420_oxidored PF03807.6 −34.5 NADP oxidoreductase coenzyme F420-
    dependent
    FAR1 PF03101.4 0 FAR1 family
    FA_hydroxylase PF04116.2 −64.1 Fatty acid hydroxylase
    FBPase PF00316.10 −170.3 Fructose-1-6-bisphosphatase
    FH2 PF02181.13 −98.3 Formin Homology 2 Domain
    GDC-P PF02347.5 −306.2 Glycine cleavage system P-protein
    GH3 PF03321.3 −336 GH3 auxin-responsive promoter
    GTP_cyclohydro2 PF00925.11 −49 GTP cyclohydrolase II
    Gln-synt_C PF00120.14 −124 Glutamine synthetase, catalytic domain
    Gln-synt_N PF03951.9 9 Glutamine synthetase, beta-Grasp domain
    Glyco_transf_8 PF01501.9 −43.2 Glycosyl transferase family 8
    Glycolytic PF00274.9 −174.5 Fructose-bisphosphate aldolase class-I
    Gp_dh_C PF02800.9 −64.1 Glyceraldehyde 3-phosphate dehydrogenase,
    C-terminal domain
    Gp_dh_N PF00044.12 −74.2 Glyceraldehyde 3-phosphate dehydrogenase,
    NAD binding domain
    HD PF01966.11 18 HD domain
    HLH PF00010.15 8.2 Helix-loop-helix DNA-binding domain
    HTH_11 PF08279.1 11.3 HTH domain
    Histone PF00125.13 17.4 Core histone H2A/H2B/H3/H4
    Kelch_1 PF01344.14 7.8 Kelch motif
    Kelch_2 PF07646.4 14 Kelch motif
    L51_S25_CI-B8 PF05047.5 25 Mitochondrial ribosomal protein L51/S25/
    CI-B8 domain
    LEA_3 PF03242.3 25 Late embryogenesis abundant protein
    LMBR1 PF04791.5 −116.3 LMBR1-like membrane protein
    LRRNT_2 PF08263.2 18.6 Leucine rich repeat N-terminal domain
    LRR_1 PF00560.21 7.7 Leucine Rich Repeat
    LSM PF01423.12 13.7 LSM domain
    Lung_7-TM_R PF06814.3 25 Lung seven transmembrane receptor
    MAP65_ASE1 PF03999.2 −134.8 Microtubule associated protein (MAP65/ASE1
    family)
    MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
    MMR_HSR1 PF01926.11 31.2 GTPase of unknown function
    MannoseP_isomer PF01050.8 −70 Mannose-6-phosphate isomerase
    Metalloenzyme PF01676.7 −14.4 Metalloenzyme superfamily
    Metallophos PF00149.17 22 Calcineurin-like phosphoesterase
    Methyltransf_11 PF08241.1 17.1 Methyltransferase domain
    Methyltransf_12 PF08242.1 21.4 Methyltransferase domain
    Mit_rib_S27 PF08293.1 25 Mitochondrial ribosomal subunit S27
    MtN3_slv PF03083.5 −0.8 MtN3/saliva family
    NAD_Gly3P_dh_N PF01210.12 −44 NAD-dependent glycerol-3-phosphate
    dehydrogenase N-terminus
    NAD_binding_1 PF00175.10 −3.9 Oxidoreductase NAD-binding domain
    NAF PF03822.4 4.5 NAF domain
    NDK PF00334.9 −59.9 Nucleoside diphosphate kinase
    NPH3 PF03000.4 25 NPH3 family
    NTP_transferase PF00483.12 −90.5 Nucleotidyl transferase
    Na_H_Exchanger PF00999.10 −67.9 Sodium/hydrogen exchanger family
    ORMDL PF04061.4 25 ORMDL family
    Oleosin PF01277.7 −27 Oleosin
    PAN_2 PF08276.2 −4.9 PAN-like domain
    PAP_fibrillin PF04755.2 25 PAP_fibrillin
    PBD PF00786.17 12.2 P21-Rho-binding domain
    PCI PF01399.15 25 PCI domain
    PEP-utilizers PF00391.12 10 PEP-utilising enzyme, mobile domain
    PGAM PF00300.12 −3 Phosphoglycerate mutase family
    PK PF00224.10 −244 Pyruvate kinase, barrel domain
    PK_C PF02887.5 −44 Pyruvate kinase, alpha/beta domain
    PPR PF01535.11 0 PPR repeat
    PTR2 PF00854.12 −50 POT family
    Pantoate_transf PF02548.5 −93 Ketopantoate hydroxymethyltransferase
    Peptidase_S10 PF00450.11 −198 Serine carboxypeptidase
    PfkB PF00294.13 −67.8 pfkB family carbohydrate kinase
    Phosphorylase PF00343.9 −601.1 Carbohydrate phosphorylase
    Pkinase PF00069.14 −70.8 Protein kinase domain
    Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
    Prenylcys_lyase PF07156.3 −164.1 Prenylcysteine lyase
    PurA PF04845.3 25 PurA ssDNA and RNA-binding protein
    RMMBL PF07521.1 18.5 RNA-metabolising metallo-beta-lactamase
    Response_reg PF00072.12 4 Response regulator receiver domain
    Rhodanese PF00581.9 25 Rhodanese-like domain
    Rib_5-P_isom_A PF06026.4 25 Ribose 5-phosphate isomerase A
    (phosphoriboisomerase A)
    Ribonuclease_T2 PF00445.8 −53 Ribonuclease T2 family
    Ribosomal_S8e PF01201.11 25 Ribosomal protein S8e
    Rotamase PF00639.10 4 PPIC-type PPIASE domain
    RrnaAD PF00398.9 −73.3 Ribosomal RNA adenine dimethylase
    SAM_1 PF00536.18 11.3 SAM domain (Sterile alpha motif)
    SMC_N PF02463.8 −95.8 RecF/RecN/SMC N terminal domain
    SNF5 PF04855.3 25 SNF5/SMARCB1/INI1
    SWIB PF02201.9 −7 SWIB/MDM2 domain
    SWIM PF04434.7 10 SWIM zinc finger
    S_locus_glycop PF00954.11 −12.7 S-locus glycoprotein family
    Sad1_UNC PF07738.2 −20.4 Sad1/UNC-like C-terminal
    Sterol_MT_C PF08498.1 30.5 Sterol methyltransferase C-terminal
    Subtilisin_N PF05922.6 26.1 Subtilisin N-terminal Region
    Sugar_tr PF00083.13 −85 Sugar (and other) transporter
    TFIIA PF03153.4 25 Transcription factor IIA, alpha/beta subunit
    TFIIS_C PF01096.9 15 Transcription factor S-II (TFIIS)
    TFIIS_M PF07500.3 7.4 Transcription factor S-II (TFIIS), central
    domain
    TIM PF00121.8 −97 Triosephosphate isomerase
    TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
    TPR_2 PF07719.5 20.1 Tetratricopeptide repeat
    TPT PF03151.7 −15.3 Triose-phosphate Transporter family
    Thioredoxin PF00085.9 −25.7 Thioredoxin
    Tim17 PF02466.8 2.7 Tim17/Tim22/Tim23 family
    TrkA_N PF02254.7 4.7 TrkA-N domain
    Tryp_alpha_amyl PF00234.10 −4 Protease inhibitor/seed storage/LTP family
    UPF0261 PF06792.1 25 Uncharacterised protein family (UPF0261)
    UQ_con PF00179.16 −30 Ubiquitin-conjugating enzyme
    Usp PF00582.16 36.1 Universal stress protein family
    WD40 PF00400.20 21.5 WD domain, G-beta repeat
    adh_short PF00106.14 −17 short chain dehydrogenase
    elF-4B PF06273.1 −205.7 Plant specific eukaryotic initiation factor 4B
    iPGM_N PF06415.3 −263.4 BPG-independent PGAM N-terminus
    (iPGM_N)
    p450 PF00067.11 −105 Cytochrome P450
    zf-AN1 PF01428.6 0 AN1-like Zinc finger
    zf-C3HC4 PF00097.13 16.9 Zinc finger, C3HC4 type (RING finger)
    zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
    similar)
    zf-CCHC PF00098.12 17.9 Zinc knuckle
    zf-CSL PF05207.3 2.9 CSL zinc finger
    • Example 5 Plasmid Contraction 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 1 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. Corn Transformation Vector
  • Elements of an exemplary common expression vector, pMON93093 are illustrated in Table 18. The exemplary base vector which is especially useful for corn transformation is illustrated in FIG. 2 and assembled using technology known in the art. The DNA of interest are inserted in a expression vector at the insertion site between the intron1 of rice act 1 gene and the termination sequence of PinII gene.
  • TABLE 18
    pMON93093
    Coordinates of
    SEQ ID NO:
    function name annotation 19940
    Agro B-AGRtu.right border Agro right border 11364-11720
    transforamtion sequence, essential for
    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- Synthetic CP4 coding 4466-5833
    CP4.nat region with dicot
    preferred codon usage.
    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.
    Agro B-AGRtu.left border Agro left border 6168-6609
    transformation sequence, essential for
    transfer of T-DNA.
    Maintenance OR-Ec.oriV-RK2 The vegetative origin of 6696-7092
    in E. coli 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. Soybean Transformation Vector
  • Plasmids for use in transformation of soybean are also prepared. Elements of an exemplary common expression vector plasmid pMON82053 are shown in Table 19 below. This exemplary soybean or canola transformation base vector illustrated in FIG. 3 is assembled using the technology known in the art. Recombinant DNA of interest, i.e. each DNA identified in Table 1 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 19
    pMON82053
    Coordinates of
    SEQ ID NO:
    function name annotation 19941
    Agro B-AGRtu.left Agro left border sequence, essential 6144-6585
    transforamtion border for transfer of T-DNA.
    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;
    Agro B-AGRtu.right Agro right border sequence, essential 1033-1389
    transformation border for transfer of T-DNA.
    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 primer 3961-4152
    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. Cotton Transformation Vector
  • Plasmids for use in transformation of cotton are also prepared. Elements of an exemplary common expression vector plasmid pMON99053 are shown in Table 20 below and FIG. 4. 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 1 is amplified by PCR prior to insertion into the insertion site within the gene of interest expression cassette of one of the base.
  • TABLE 20
    Coordinates
    of SEQ ID
    function name annotation NO: 19942
    Agro B-AGRtu.right border Agro right border sequence, 11364-11720
    transforamtion essential for transfer of T-
    DNA.
    Gene of interest Exp-CaMV.35S- Enhanced version of the 35S 7794-8497
    expression enh + ph.DnaK RNA promoter from CaMV
    cassette plus the petunia hsp70 5′
    untranslated region
    T-Ps.RbcS2-E9 The 3′ non-translated region  67-699
    of the pea RbcS2 gene which
    functions to direct
    polyadenylation of the
    mRNA.
    Plant selectable Exp-CaMV.35S Promoter and 5′ untranslated  730-1053
    marker region of the 35S RNA from
    expression CaMV
    cassette CR-Ec.nptII-Tn5 Neomycin 1087-1881
    Phosphotransferase II gene
    that confers resistance to
    neomycin and kanamycin
    T-AGRtu.nos A 3′ non-translated region of 1913-2165
    the nopaline synthase gene of
    Agrobacterium tumefaciens
    Ti plasmid which functions to
    direct polyadenylation of the
    mRNA.
    Agro B-AGRtu.left border Agro left border sequence, 2211-2652
    transformation essential for transfer of T-
    DNA.
    Maintenance in OR-Ec.oriV-RK2 The vegetative origin of 2739-3135
    E. coli replication from plasmid
    RK2.
    CR-Ec.rop Coding region for repressor 4644-4835
    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 5263-5851
    replication from the E. coli
    plasmid ColE1.
    P-Ec.aadA-SPC/STR romoter for Tn7 6382-6423
    adenylyltransferase
    (AAD(3″))
    CR-Ec.aadA-SPC/STR Coding region for Tn7 6424-7212
    adenylyltransferase
    (AAD(3″)) conferring
    spectinomycin and
    streptomycin resistance.
    T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 7213-7270
    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 improved 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 1 by Agrobacterium-mediated transformation using the corn transformation vectors pMON93093 as disclosed in Example 6. 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 improved 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 improved 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 vectors pMON82053 disclosed in Example 7. 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 1.
  • 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 improved seed composition.
    • Example 8 Canola Transformation
  • 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 overnight-grown Agrobacterium cells 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 characterization 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 1. 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 as reported in Example 9.
    • Example 9 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 1 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 improved yield as compared to a control plant. Improved 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 improved 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 may 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. It is to plant multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, for example 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. Transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field. A pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events. A useful planting density is about 30,000 plants/acre. High planting density is greater than 30,000 plants/acre, preferably about 40,000 plants/acre, more preferably about 42,000 plants/acre, most preferably about 45,000 plants/acre. 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 and soybean 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 height 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 may 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 improvement (DS) 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 experiment.
  • 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+LH59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7 C 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 experiment. 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 improvement (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 21
    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 run: 1.5 minute per sample
    Typical and minimum sample Corn typical: 50 cc; minimum 30 cc
    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 10 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: 197 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 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 by Pfam, SNF5, LMBR1, TFIIS_M, TFIIS_C, or Glyco_transf—8. 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 with an amino acid sequence of SEQ ID NO:200, 201, 205, 207, 211, and 394.
  • Populations of transgenic corn plants and soybean plants prepared in Examples 6 and 7 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.

Claims (18)

1. A plant cell nucleus with stably integrated, recombinant DNA comprising a promoter that is functional in plant cells and that is operably linked to DNA from a plant, bacteria or yeast that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name selected from the group of Pfam names consisting of L51_S25_CI-B8, iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap, Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RmaAD, NAF, BolA, Pkinase, C2, FA_hydroxylase, p450, Complex1—30 kDa, Histone, DUF822, PEP-utilizers, PCI, ETC_C1_NDUFA5,2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE1, FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro2, PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5, Ceramidase_alk, Ribonuclease_T2, Complex1—49 kDa, Gp_dh_C, Aldo_ket_red, zf-AN1, TFIIS_C, MFS—1, Thioredoxin, DUF1005, LEA—3, Sterol_MT_C, Gp_dh_N, TFIIS_M, PAN—2, BPL_C, DUF26, Aa_trans, ACT, ADH_N, NAD_binding—1, Auxin_inducible, B_lectin, Anti-silence, Response_reg, 14-3-3, LRRNT—2, GDC-P, zf-CCHC, NPH3, TPR—1, TFIIA, DHBP_synthase, UQ_con, TPR—2, TPT, F-box, adh_short, Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv, TIM, NDK, Pantoate_transf, Allene_ox_cyc, Cyclin_N, Methyltransf—1, CBM—20, Methyltransf—12, Rhodanese, Glycolytic, Actin, Usp, eIF-4B, Glyco_transf—8, BURP, Alpha-amylase, F420_oxidored, EGF_CA, Kelch—1, PGAM, Aminotran—1—2, Kelch—2, UPF0261, CoA_binding, DUF868, Peptidase_S10, Lung—7-TM_R, Oleosin, Sad1_UNC, Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase, Subtilisin_N, SAM—1, DUF298, ESCRT-III, DNA_pol_E_B, Aminotran—3, NAD_Gly3P_dh_N, Gln-synt_N, MMR_HSR1, DUF588, zf-CCCH, DnaJ, Pkinase_Tyr, Cupin—2, LRR—1, Cupin—3, zf-CSL, FAR1, HD, FH2, APC8, PTR2, MannoseP_isomer, Rib—5-P_isom_A, DUF1336, Phosphorylase, DUF1191, Asp, Mit_rib_S27, PAP_fibrillin, DUF1195, Aldedh, zf-C3HC4, PPR, PK, PurA, RMMBL, HTH—11, Tim17, and PBD wherein said Pfam gathering cutoff for said protein domain families are stated in Table 17; wherein said plant cell nucleus is selected by screening a population of transgenic plants that have said recombinant DNA and express said protein for an enhanced trait as compared to control plants that do not have said recombinant DNA in their nuclei; and wherein said enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced resistance to salt exposure, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
2. The plant cell nucleus of claim 1 wherein said protein has an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 198 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 394 and homologs thereof listed in Table 2.
3. The plant cell nucleus of claim 1 wherein said protein is selected from the group of proteins identified in Table 1.
4. A plant cell nucleus with stably integrated, recombinant DNA to suppress the level of an endogenous protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by Pfam name in the group of Pfam names consisting of SNF5, LMBR1, TFIIS_M, TFIIS_C, and Glyco_transf—8, wherein the Pfam gathering cutoff for said protein domain families is stated in Table 17 wherein said plant cell nucleus is selected by screening a population of transgenic plants with said recombinant DNA and have the level of said endogenous protein suppressed for an enhanced trait as compared to control plants that do not have said recombinant DNA; and wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced resistance to salt exposure, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
5. The plant cell nucleus of claim 1 or 4 further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell.
6. The plant cell nucleus of claim 5 wherein the agent of said herbicide is a glyphosate, dicamba, or glufosinate compound.
7. A transgenic plant cell or plant comprising a plurality of plant cells with a plant cell nucleus of claim 1 or 4.
8. The transgenic plant cell or plant of claim 7 which is homozygous for said recombinant DNA.
9. A transgenic seed comprising a plurality of plant cells with a plant cell nucleus of claim 1 or 4.
10. The transgenic seed of claim 9 from a corn, soybean, cotton, canola, alfalfa, wheat or rice plant.
11. The transgenic corn seed of claim 10 wherein said seed can produce corn plants that are resistant to disease from the Mal de Rio Cuarto virus or the Puccina sorghi fungus or both.
12. A transgenic pollen grain comprising a haploid derivative of a plant cell nucleus of claim 1 or 4.
13. A method 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 a nucleus of claim 1 or 4, wherein said method for manufacturing said transgenic seed comprising:
(a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population can exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not express the recombinant DNA,
(b) selecting from said population one or more plants that exhibit said trait at a level greater than the level that said trait is exhibited in control plants,
(c) verifying that said recombinant DNA is stably integrated in said selected plants,
(d) analyzing tissue of said selected plant to determine the production or suppression of a protein having the function of a protein encoded by nucleotides having a sequence selected from the group consisting of one of SEQ ID NO:198-394; and
(e) collecting seed from said selected plant.
14. The method of claim 14 wherein plants in said population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells, and wherein said selecting is effected by treating said population with said herbicide.
15. The method of claim 14 wherein said herbicide comprises a glyphosate, dicamba, or glufosinate compound.
16. The method of claim 15 wherein said selecting is effected by identifying plants with said enhanced trait.
17. The method of claim 16 wherein said seed is corn, soybean, cotton, alfalfa, wheat or rice seed.
18. A method of producing hybrid corn seed comprising:
(a) acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA in a nucleus of claim 1 or 4;
(b) producing corn plants from said hybrid corn seed, wherein 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 a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA;
(c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide;
(d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants;
(e) repeating steps (c) and (d) at least once to produce an inbred corn line;
(f) crossing said inbred corn line with a second corn line to produce hybrid seed.
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