Transgenic plants with enhanced agronomic traits   

This application claims benefit under 35USC §119(e) of U.S. provisional application Ser. No. 60/958,909, filed Jul. 10, 2007 herein incorporated by reference in its entirety.

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 text file named 38-21(54973)A_segListing.txt, which is 105,877,504 bytes (measured in MS-WINDOWS), were created on Jul. 10, 2008 and are herein incorporated by reference.

Two copies of the Computer Program Listing (Copy 1 and Copy 2) and a computer readable form (CRF) containing folders hmmer-2.3.2 and 226pfamDir, all on CD-Rs are 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 226pfamDir contains 226 Pfam Hidden Markov Models. Both folders were created on CD-R on Jul. 9, 2007, having a total size of 19,894,272 bytes (measured in MS-WINDOWS).

FIELD OF THE INVENTION

Disclosed herein are recombinant DNA useful for providing enhanced traits to transgenic plants, seeds, pollen, plant cells and plant nucleui of such transgenic plants, methods of making and using such recombinant DNA, plants, seeds, pollen, plant cells and plant nuclei. Also disclosed are methods of producing hybrid seed comprising such recombinant DNA.

SUMMARY

This invention employs recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic traits to the transgenic plants. 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 a DNA segment that encodes a protein. In some embodiments of the invention, such protein defined by protein domains e.g. a “Pfam domain module” (as defined herein below) from the group of Pfam domain modules identified in Table 11. In other embodiments of the invention, e.g. where a Pfam domain module is not available, such protein is defined a consensus amino acid sequence of an encoded protein that is targeted for production e.g. a protein having amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of SEQ ID NO: 30526 through SEQ ID NO: 30550. In more specific embodiments of the invention the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 2 and their homologs identified in Table 8.

Other aspects of the invention are specifically directed to plant cell nuclei and transgenic cells comprising the recombinant DNA construct of the invention, transgenic plants comprising a plurality of such plant cells, progeny transgenic seed, embryo and transgenic pollen from such plants. Such transgenic plants are selected from a population of transgenic plants regenerated from plant cells transformed with recombinant DNA construct and expressed the protein by screening transgenic plants in the population for an enhanced trait as compared to control plants that do not have the recombinant DNA construct, where the enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

In yet another aspect of the invention the plant cell nuclei, plant cells, transgenic plants, seeds, and pollen further comprise recombinant 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 such herbicide is a glyphosate, dicamba, or glufosinate compound.

Yet other aspects of the invention provide 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.

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 construct provided by herein. More specifically the method comprises (a) screening a population of plants for an enhanced trait and a recombinant DNA construct of the invention, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants which do not express the recombinant DNA, (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants, and (c) collecting seed from a selected plant. The method further comprises (d) verifying that the recombinant DNA construct is stably integrated in said selected plants, and (e) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein selected from SEQ ID NO: 299 through SEQ ID NO: 30468. In one aspect of the invention the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to a herbicide applied at levels that are lethal to wild type plant cells and the selecting is affected 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, canola, alfalfa, wheat or rice seed.

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 stably-integrated, recombinant DNA construct comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA that encodes a protein provided by the invention. The methods further comprise producing corn plants from the 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 the recombinant DNA construct, and a fraction of the plants produced from said hybrid corn seed has none of the recombinant DNA construct; selecting corn plants which are homozygous and hemizygous for the recombinant DNA construct by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting the seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a consensus amino acid sequence of SEQ ID NO: 301 and its homologs.

FIGS. 2-4 are plasmid maps.

DETAILED DESCRIPTION

In the attached sequence listing:

SEQ ID NO:1-298 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: 299-596 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequences 1-298;

SEQ ID NO: 597-30468 are amino acid sequences of homologous proteins;

SEQ ID NO: 30469-30520 are nucleotide sequences of the elements in base plasmid vectors

SEQ ID NO: 30521 is a nucleotide sequence of a base plasmid vector useful for corn transformation;

SEQ ID NO: 30522 is a nucleotide sequence of a base plasmid vector useful for soybean and canola transformation;

SEQ ID NO: 30523 is a nucleotide sequence of a base plasmid vector useful for cotton transformation;

SEQ ID NO: 30524 is a nucleotide sequence of a Sphas1 promoter from soybean;

SEQ ID NO: 30525 is a nucleotide sequence of a Sphas1 leader from soybean.

SEQ ID NO: 30526-30550 are consensus sequences.

Table 1 lists the protein SEQ ID NOs and their corresponding consensus SEQ ID NOs.

TABLE 1 Consensus Gene ID PEP SEQ ID NO SEQ ID NO PHE0006007_18714 301 30526 PHE0006460_15962 453 30527 PHE0006657_16192 334 30528 PHE0006712_16273 415 30529 PHE0006859_16873 376 30530 PHE0006907_16797 594 30531 PHE0006936_16828 348 30532 PHE0006969_16871 428 30533 PHE0007578_17852 446 30534 PHE0007650_18175 358 30535 PHE0008172_18471 397 30536 PHE0008308_18841 392 30537 PHE0008340_19155 410 30538 PHE0008422_18842 412 30539 PHE0008698_24421 364 30540 PHE0010197_21429 569 30541 PHE0010201_21433 576 30542 PHE0010201_21768 574 30543 PHE0010223_21491 545 30544 PHE0010397_21762 538 30545 PHE0010398_21763 539 30546 PHE0010615_22400 510 30547 PHE0010838_22702 575 30548 PHE0011447_23660 586 30549 PHE0012170_24424 572 30550

As used herein a “plant cell” means a plant cell that is transformed with stably-integrated, non-natural, recombinant DNA construct, e.g. by Agrobacterium-mediated transformation or by bombardment using microparticles coated with recombinant DNA construct or other means. A plant cell of this invention can be an originally-transformed plant cell that exists as a microorganism or as a progeny plant cell that is regenerated into differentiated tissue, e.g. into a transgenic plant with stably-integrated, non-natural recombinant DNA, or seed or pollen derived from a progeny transgenic plant.

As used herein a “transgenic plant” means a plant whose genome has been altered by the stable integration of recombinant DNA construct. A transgenic plant includes a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant.

As used herein “recombinant DNA” means DNA which has been a genetically engineered and constructed outside of a cell including DNA containing naturally occurring DNA or cDNA or synthetic DNA.

As used herein “consensus sequence” means an artificial sequence of amino acids in a conserved region of an alignment of amino acid sequences of homologous proteins, e.g. as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.

As used herein a “homolog” means a protein in a group of proteins that perform the same biological function, e.g. proteins that belong to the same Pfam protein family and that provide a common enhanced trait in transgenic plants of this invention. Homologs are expressed by homologous genes. Homologous genes include naturally occurring alleles and artificially-created variants. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, a polynucleotide useful in the present invention may have any base sequence that has been changed from SEQ ID NO:1 through SEQ ID NO: 298 substitution in accordance with degeneracy of the genetic code. Homologs are proteins that, when optimally aligned, have at least 60% identity, more preferably about 70% or higher, more preferably at least 80% and even more preferably at least 90% identity over the lull length of a protein identified as being associated with imparting an enhanced trait when expressed in plant cells. Homologs include proteins with an amino acid sequence that has at least 90% identity to a consensus amino acid sequence of proteins and homologs disclosed herein.

Homologs are identified by comparison of amino acid sequence, e.g. manually or by use of a computer-based tool using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. A local sequence alignment program, e.g. BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. 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 query is used in the present invention to filter hit sequences with significant E-values for ortholog identification. The reciprocal query entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal query\'s best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. A further aspect of the invention comprises functional homolog proteins that differ in one or more amino acids from those of disclosed protein as the result of conservative amino acid substitutions, for example substitutions are among: acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; basic (positively charged) amino acids such as arginine, histidine, and lysine; neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; amino acids having aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; amino acids having aliphatic-hydroxyl side chains such as serine and threonine; amino acids having amide-containing side chains such as asparagine and glutamine; amino acids having aromatic side chains such as phenylalanine, tyrosine, and tryptophan; amino acids having basic side chains such as lysine, arginine, and histidine; amino acids having sulfur-containing side chains such as cysteine and methionine; naturally conservative amino acids such as valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the homologs encoded by DNA useful in the transgenic plants of the invention are those proteins that differ from a disclosed protein as the result of deletion or insertion of one or more amino acids in a native sequence.

“Percent identity” describes the extent to which the sequences of DNA or protein segments are invariant throughout a window of alignment of nucleotide or amino acid sequences. An “identity fraction” for a sequence aligned with a reference sequence is the number of identical components which are shared by the sequences, divided by a length of the window of alignment, wherein the length does not include gaps introduced by an alignment algorithm. “Percent identity” (“% identity”) is the identity fraction times 100. The alignment algorithm is preferably a local alignment algorithm, such as BLASTp. As used herein, sequences are “aligned” when the alignment produced by BLASTp has a minimal e-value.

“Pfam” database is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 19.0 (December 2005) contains alignments and models for 8183 protein families and is based on the Swissprot 47.0 and SP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. The Pfam database is currently maintained and updated by the Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein\'s function. Profile hidden Markov models (profile HMMs) built from the protein family alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low.

Protein domains are identified by querying the amino acid sequence of a protein against Hidden Markov Models which characterize protein family domains (“Pfam domains”) using HMMER software, a current version of which is provided in the appended computer listing. A protein domain meeting the gathering cutoff for the alignment of a particular Pfam domain is considered to contain the Pfam domain.

A “Pfam domain module” is a representation of Pfam domains in a protein, in order from N terminus to C terminus. In a Pfam domain module individual Pfam domains are separated by double colons “::”. The order and copy number of the Pfam domains from N to C terminus are attributes of a Pfam domain module. Although the copy number of repetitive domains is important, varying copy number often enables a similar function. Thus, a Pfam domain module with multiple copies of a domain should define an equivalent Pfam domain module with variance in the number of multiple copies. A Pfam domain module is not specific for distance between adjacent domains, but contemplates natural distances and variations in distance that provide equivalent function. The Pfam database contains both narrowly- and broadly-defined domains, leading to identification of overlapping domains on some proteins. A Pfam domain module is characterized by non-overlapping domains. Where there is overlap, the domain having a function that is more closely associated with the function of the protein (based on the E value of the Pfam match) is selected.

Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins with the same Pfam domain module are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Models which characterizes the Pfam domains using HMMER software. Candidate proteins meeting the same Pfam domain module are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein with a common Pfam domain module for recombinant DNA in the plant cells of this invention are also included in the appended computer listing.

The HMMER software and Pfam databases are version 19.0 and were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 299 through SEQ ID NO: 596. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 14 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 modules for use in this invention, as more specifically disclosed below, are zf-CCCH, PALP, GAF::HisKA::HATPase_c, TPR—2::TPR—1::TPR—2::TPR—1::TPR—4::TPR—2::TPR—1, efhand::efhand, Spermine_synth, ELFV_dehydrog_N::ELFV_dehydrog, PFK, PAS—3::PAS—3::Pkinase, S1, GDC-P, SWIM, B12-binding::Radical_SAM, S1, LRR—1::LRR—1::LRR—1::LRR—1::LRR—1:Chloroa_b-bind, LRRNT—2::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LR WD40::WD40, YABBY, Ldh—1_N::Ldh—1_C, Sina, Na_H_antiport—1, ParBc, YABBY, Histone, Fe_bilin_red, Tryp_alpha_amyl, Pyr_redox—2::Thioredoxin, E2F_TDP, CN_hydrolase, YDG_SRA::Pre-SET:: SET, APC8::TPR—1::TPR—1::TPR—1, Ras, tRNA_anti::tRNA-synt—2, Auxin_inducible, PGI, S1, Chloroa_b-bind, Bac_globin, Glyco_hydro—17, MGS, Spermine_synth, LRRNT—2::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::Pkinase, Aa_trans, Gln-synt_N::Gln-synt_C, SAP18, TPP_enzyme_N::TPP_enzyme_M::TPP_enzyme_C, zf-C3HC4, eIF-1a, RPE65, PBP, Pkinase, AA_permease, F-box::LRR—1::LRR—2, zf-CCCH::zf-CCCH, Lactamase_B::Flavodoxin—1::Flavin_Reduct, Bac_globin, DSPc, adh_short, Tim17, Oxidored_molyb::Mo-co_dimer:Cyt-b5::FAD_binding—6::NAD_binding—1, ubiquitin::ubiquitin::ubiquitin::ubiquitin, MBD, CXC::CXC, HSF_DNA-bind, Spermine_synth, AP2, Peptidase_S10, PALP, EIN3, Gln-synt_N::Gln-synt_C, 2OG-FeII_Oxy, Glyco_hydro—9, GDC-P, B3, PTPA, Acyltransferase, Isochorismatase, FMO-like, Molybdop_Fe4S4::Molybdopterin::Molydop_binding::Fer2_BFD, Lir1, Prismane, Fer2, DEAD::Helicase_C, Molybdop_Fe4S4::Molybdopterin::Molydop_binding::Fer2_BFD, KNOX1::KNOX2::ELK, Glyoxalase, Sad1_UNC, Bac_globin, VPS28, PP2C, Pkinase::efhand::efhand::efhand, LEA—3, Peptidase_S10, Pkinase, CBFDNFYB_HMF, Gln-synt_N::Gln-synt_C, Pyr_redox—2::Fer2_BFD::NIR_SIR_ferr::NIR_SIR, NUDIX::NUDIX, FAD_binding—3, GST_N::GSTS, SAM_decarbox, Acyltransferase, NTP_transferase, G-patch, 2OG-FeIl_Oxy, Gln-synt_N::Gln-synt_C, AAA::Vps4_C, Histone, Pkinase, TPR—1, F-box::Kelch—1::Kelch—1, Spermine_synth, Bac_globin, Bac_gldobin, zf-UBR, Homeobox::HALZ, Whirly, NAD_binding—1, PTR2, EIN3, 4HBT, adh_short, 2OG-FeII_Oxy, P-II, Myb_DNA-binding::Myb_DNA-binding, DAGK_cat, AP2, MFS1, Chloroab-bind, DUF716, zf-D of, CCT, Homeobox::HALZ, Histone, 2OG-FeII_Oxy, Globin, Pyr_redox 2::Fer2_BFD::NIR_SIR_ferr::NIR_SIR, Whirly, PsbP, bZIP—1, LRRNT—2::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LR R—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::Pkinase_Tyr, Phi—1, BURP, Sterol_desat, DSPc, SNF5, Acyltransferase, GATase—2::Asn_synthase, adh_short, Homeobox:: START, Pkinase, ParBc, SOUL, DNA_photolyase::FAD_binding—7, Pkinase_Tyr, NAD_Gly3P_dh_N::NAD_Gly3P_dh_C, Na_H_Exchanger, peroxidase, Oxidored_molyb::Mo-co_dimen:Cyt-b5::FAD_binding—6::NAD_binding—1, Hexokinase—1::Hexokinase—2, DUF716, S10_plectin, Thi4, p450, CCT, adh_short, PSI_PSAK, DUF640, Thioredoxin, Globin, Ank::Pkinase, DAGAT, RPE65, Ank::Pkinase, GSHPx, Gln-synt_N::Gln-synt_C, MtN3_sly::MtN3_sly, Allene_ox_cyc, IGPD, MBD, CorA, Response_reg, Histone, AAA, Ribosomal_L10e, Pkinase, DUF26::DUF26::Pkinase, p450, mTERF, AA_kinase, PBP, GUN4, Lactamase_B::Flavodoxin—1::Rubredoxin, C2, RRM—1::RRM—1, Histone, Alpha-amylase, HLH, Thioredoxin, Histone_HNS, Myb_DNA-binding::Myb_DNA-binding, Cytochrom_C552, AP2::AF\'2, MtN3_sly::MtN3_sly, SHMT, ParBc, Mit_rib_S27, Ribosomal_S2, KNOX1::KNOX2::ELK, MFS—1, Glyco_transf—5::Glycos_transf—1, Cellulase, Ribosomal_L10e, Spermine_synth, Glyco_hydro—2_N::Glyco_hydro—2::Glyco_hydro—2_C, TP_methylase, AP2::AP2, Histone, Response_reg::CCT, Histone_HNS, DUF1716, p450, GATA, Pkinase, Sugar_tr, Aa_trans, Pribosyltran, Ribosomal_L10e, HLH, PMSR, DnaJ::DnaJ_CXXCXGXG::DnaJ_C, DUF1005, Glyco_transf—5::Glycos_transf—1, Spermine_synth, S1::EIF—2_alpha, RGS, Na_sulph_symp, S1, MtN3_sly::MtN3_sly, Lactamase_B::Flavin_Reduct, LRRNT—2::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LR R—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::LRR—1::Pkinase, Chloroa_b-bind, PTR2, Agglutinin, PLATZ, NPH3, Auxin_inducible, PTR2, GAF::HisKA::Response_reg, PsbQ, GSH_synth_ATP, GATase—2::Asn_synthase, PHP, FtsJ, DUF6::TPT, Proteasome, PsbW—2, Glyco_hydro—9, NAD_binding—2::6 PGD, S1::EIF—2_alpha, Homeobox::START::MEKHLA, S1, Isoamylase_N::Alpha-amylase, E2F_TDP, and Rieske::PaO, for which the databases are included in the appended computer listing.

As used herein “promoter” means regulatory DNA for initializing transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell, e.g. is it well known that Agrobacterium promoters are functional in plant cells. Thus, plant promoters include promoter DNA obtained from plants, plant viruses and bacteria such as Agrobacterium and Bradyrhizobium bacteria. 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 that 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 “operably linked” means the association of two or more DNA fragments in a DNA construct so that the function of one, e.g. protein-encoding DNA, is controlled by the other, e.g. a promoter.

As used herein “expressed” means produced, e.g. a protein is expressed in a plant cell when its cognate DNA is transcribed to mRNA that is translated to the protein.