Patent Application
20160264980
Three copies of the a sequence listing (Copy 1 and Copy 2 and a computer readable form), in a text file named “3126.009US3 seq listing in text (1539270×7ADA8).TXT” which is 93,526,016 bytes (measured in MS-WINDOWS) are provided on separate CD-ROMs which were created on Jan. 29, 2016 and are herein incorporated by reference.
Two copies of Computer Program Listing (Copy 1 and Copy 2) containing folders “hmmer-2.3.2” and “241pfam” are provided on separate CD-ROMs that were created on Jan. 22, 2016, and have a total file size of 20,899,840 bytes (measured in MS-WINDOWS). The “hmmer-2.3.2” folder contains the source code and other associated ASCII files for implementing the HMMer software for Pfam analysis; the “241pfam” folder contains ASCII files of 241 Pfam Hidden Markov Models; all of which are incorporated herein by reference in their entirety.
Two copies of Table 7 (Copy 1 and Copy 2) are provided on CD-ROMs that were created on Jan. 25, 2016 and contain the file named “38-21_52054D_table7_convertedFromPDF.txt” which is 335,872 bytes (measured in MS-WINDOWS) and which comprise 89 pages when viewed in MS Word®, are herein incorporated by reference.
Disclosed herein are inventions in the field of plant genetics and developmental biology. More specifically, the present inventions provide plant cells with recombinant DNA for providing an enhanced trait in a transgenic plant, plants comprising such cells, seed and pollen derived from such plants, methods of making and using such cells, plants, seeds and pollen.
This invention provides plant cell nuclei with recombinant DNA that imparts enhanced agronomic traits in transgenic plants having the nuclei in their cells, e.g. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil. Such recombinant DNA in a plant cell nucleus of this invention is provided in as a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein or to DNA that results in gene suppression. Such DNA in the construct is sometimes defined by protein domains of an encoded protein targeted for production or suppression, e.g. a “Pfam domain module” (as defined herein below) from the group of Pfam domain modules identified in Table 21 (page 94). Alternatively, e.g. where a Pfam domain module is not available, such DNA in the construct is defined a consensus amino acid sequence of an encoded protein that is targeted for production 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: 27377 through SEQ ID NO: 27426. Alternatively, in other cases where neither a Pfam domain module nor a consensus amino acid sequence is available, such DNA in the construct is defined by the sequence of a specific encoded and or its homologue proteins.
Other aspects of the invention are specifically directed to transgenic plant cells comprising the recombinant DNA 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 and expressed the protein by screening transgenic plants in the population for an enhanced trait as compared to control plants that do not have said recombinant DNA, 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 cells, 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 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 in the nucleus of the plant cells. 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 and (c) collecting seed from a selected plant. Such method further comprises steps (a) verifying that the recombinant DNA is stably integrated in said selected plants; and (b) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by a recombinant DNA with a sequence of one of SEQ ID NO: 1-614; 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 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 stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA 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 in the group of Pfam names identified in Table 22. The methods further comprise 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; 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 plant cells of the invention by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue. Yet another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a plant cell of this invention.
Still other aspects of this invention relate to 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.
In the attached sequence listing:
SEQ ID NO:1-614 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: 615-1228 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequences 1-614;
SEQ ID NO: 1229-27373 are amino acid sequences of homologous proteins;
SEQ ID NO: 27374 is a nucleotide sequence of a base plasmid vector useful for corn transformation;
SEQ ID NO: 27375 is a nucleotide sequence of a base plasmid vector useful for soybean and canola transformation;
SEQ ID NO: 27376 is a nucleotide sequence of a base plasmid vector useful for cotton transformation;
SEQ ID NO: 27377-27426 are consensus sequences.
As used herein a “plant cell” means a plant cell that is transformed with stably-integrated, non-natural, recombinant DNA, e.g. by Agrobacterium-mediated transformation or by bombardment using microparticles coated with recombinant DNA 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. 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 useful polynucleotide may have base sequence changes from SEQ ID NO:1 through SEQ ID NO: 614 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, e.g. 95 to 98% identity, over the full 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 have at least 90% identity, e.g. at least 95 to 98% 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 ut more amino acids in a native sequence.
As used herein, “percent identity” means the extent to which two optimally aligned DNA or protein segments are invariant throughout one or more windows of alignment of components, for example nucleotide sequence or amino acid sequence, allowing for gaps to account to insertions and deletions. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by sequences of the two aligned segments divided by the total number of sequence components in the aligned parts of the reference segment over window(s) 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.
The “Pfam” database is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 19.0 (December 2005) contains alignments and models for 8183 protein families and is based on the Swissprot 47.0 and SP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. The Pfam database is currently maintained and updated by the Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the protein family alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low.
A “Pfam domain module” is a representation of Pfam domains in a protein, in order from N terminus to C terminus. In a Pfam domain module individual Pfam domains are separated by double colons “::”. The order and copy number of the Pfam domains from N to C terminus are attributes of a Pfam domain module. Although the copy number of repetitive domains is important, varying copy number often enables a similar function. Thus, a Pfam domain module with multiple copies of a domain should define an equivalent Pfam domain module with variance in the number of multiple copies. A Pfam domain module is not specific for distance between adjacent domains, but contemplates natural distances and variations in distance that provide equivalent function. The Pfam database contains both narrowly- and broadly-defined domains, leading to identification of overlapping domains on some proteins. A Pfam domain module is characterized by non-overlapping domains. Where there is overlap, the domain having a function that is more closely associated with the function of the protein (based on the E value of the Pfam match) is selected.
Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins with the same Pfam domain module are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Models which characterizes the Pfam domains using HMMER software, a current version of which is provided in the appended computer listing. Candidate proteins meeting the same Pfam domain module are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein with a common Pfam domain module for recombinant DNA in the plant cells of this invention are also included in the appended computer listing. Version 19.0 of the HMMER software and Pfam databases were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 615 through SEQ ID NO: 1228. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 23 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 DUF6::DUF6, Sterol_desat, HMG_box, GAF::HisKA::HATPase_c, Sugar_tr, Mito_carr::Mito_carr::Mito_carr, RRM_1, 14-3-3, Globin, F-box::Kelch_1::Kelch_2::Kelch_1::Kelch_2::Kelch_2, Pkinase, zf-CIIY::zf-C3HC4, AUX_IAA, Cu-oxidase_3::Cu-oxidase::Cu-oxidase_2, Sigma70_r2::Sigma70_r3::Sigma70_r4, AT_hook::DUF296, Exo_endo_phos, H_PPase, Aldo_ket_red, CHASE::HisKA::HATPase_c::Response_reg, Myb_DNA-binding, AP2::AP2, Flavodoxin 2, P-11, zf-CCCH::zf-CCCH::KH_1::zf-CCCII, PSK, adh_short, Myb_DNA-binding::Myb_DNA binding, FLO_LFY, LRR_1::LRR_1::LRR_::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::Pkinase, Zein, Response_reg::Myb_DNA-binding, LEA_4, DAD, DUF6::DUF6, F-box::LRR_2, LEA_2, zf-C3HC4, 2OG-FeII_Oxy, WD40::WD40, DUF231, Cullin, CBFD_NFYB_HMF, Histone, U-box, HSF_DNA-bind, GH3, LIM::LIM, RPE65, GST_N::GST_C, IMPDH, Mlo, Copine, Rieske::PaO, ADH_N::ADH_zinc_N, PBP, F-box, Prp19::WD40::WD40::WD40, (Glycos_transf_1::S6PP, PtkB, ABA_WDS, AP2, Asp, Hydrolase, OPT, TFIIS::TFIIS_M::TFIIS_C, Peptidase_C14, TPT, NAM, SRF-TF::K-box, G-alpha, Lactamase_B, LRR_2::LRR_2, PTR2, PB1, Pkinase::Pkinase_C, S-methyl_trans, Phytochrome::HisKA::HATPase_c, Ank::Ank::Ank::Ank::Ank::zf-C3HC4, F-box::Kelch_2::Kelch_2::Kelch_1::Kelch_2, Cyclin_N, Dor1, F-box::LRR_1, BCCT, B_lectin::S_locus_glycop::PAN_2::Pkinase, SAC3_GANP, F-box::Kelch_1::Kelch_1::Kelch_1, DUF6, MFMR::bZIP_1, Skp1_POZ::Skp1, U-box::Arm::Arm::Arm, NAF1, Ribosomal_L18p, SET, F-box::LysM, Pyridoxal_dcC, PPDK_N::PEP-utilizers::PEP-utilizers_C, Transket_pyr::Transketolase_C, IPP-2, zf-B_box::zf-B_box::CCT, MFS_1, zf-Dof, RRM_::zf-CCHC, F-box::Tub, SATase_N::Hexapep::Hexapep::Hexapep, PEMT, B_lectin::PAN_2::Pkinase, Pcptidase_S10, SOH1, Methyltransf_11, bZIP_1, DXP_reductoisom::DXP_redisom_C, Flavoprotein, MatE::MatE, Homeobox::HALZ, U-box::Arm::Arm::Arm::Arm::Arm, zf-B_box::zf-B_box, Glycos_transf_1, zf-LSD1::zf-LSD1::zf-LSD1, Aldedh, Melibiase, HEAT::HEAT::HEAT::FAT::Rapamycin_bind::PI3_PI4_kinase::FATC, MtN3_slv::MtN3_sly, DUF1313, S6PP, HD-ZIP_N::Homeobox::HALZ, WRKY, FBPase_glpX, MIF4G::MIF4G_like::MIF4G_like_2, zf-B_box::CCT, FAD_binding_4, IIpt, TLC, CK_II_beta, PR_1::TPR_1::TPR_2::U-box, Response_reg, AdoHcyase_NAD, PI-PLC-X::PI-PLC-Y::C2, Pkinase::Ribonuc_2-5A, Globin::FAD_binding_6::NAD binding_1, PME1, Myb_DNA-binding::Linker_histone, LRRNT_2::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_::LRR_::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::Pkinase, Pkinase::ethand::efhand::efhand::efhand, Pescadillo N::BRCT, SPX::zf-C3HC4, AdoHcyase, zf-CCCH::zf-CCCH::zf-CCCH::zf-CCCH::zf-CCCH, SBP56, DUF850, NAS, UPF0005, Alpha-amylase::Alpha-amyl_C2, Na_H_Exchanger, PAN_1::Pkinase, F-box::Kelch_1::Kelch_1, Remorin_C, Skp1, DUF580, zf-C2H2, zf-LSD1::Peptidase_C14, Ribosomal_L10::Ribosomal_60s, Frigida, Methyltransf_11::Methyltransf_11, dCMP_cyt_deam_1, DUF914, Enolase_N::Enolase_C, p450, Cellulose_synt, Cu_bind_like, S6PP::S6PP_C, BRAP2::zf-C3HC4::zf-UBP, BIR::BIR, C1_1::DAGK_cat::DAGK_acc, PA::zf-C3HC4, DPBB_1::Pollen_allerg_1, LRRNT_2::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::LRR_1::::LRR_1::LRR_1::LRR_1::Pkinase, WD40::WD40::WD40::WD40::WD40::WD40, bZIP_2, FBPase, HLH, GRAS, SBP, Sina, Remorin_N::Remorin_C, BTB::NPH3, Glutaredoxin, AA_permease, Cyclin_N::Cyclin_C, DUF810, LRR_2, B_lectin::S_locus_glycop::PAN_2::PAN_1::Pkinase, Put_Phosphatase, DUF221, Response_reg::CCT, EMP24_GP25L, VDE, Orn_Arg_deC_N::Omn_DAP_Arg_deC, HEAT::HEAT::HEAT::HEAT::HEAT::HEAT::HEAT, PHD, UPF0041, Bromodomain, Bap31, UDPGP, Pkinase::NAF, Pirin::Pirin_C, MED7.
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.
As used herein a “control plant” means a plant that does not contain the recombinant DNA that expressed a protein that impart an enhanced trait. A control plant is to identify and select a transgenic plant that has an enhance trait. A suitable control plant can be a non-transgenic plant of the parental line used to generate a transgenic plant, i.e. devoid of recombinant DNA. A suitable control plant may in some cases be a progeny of a hemizygous transgenic plant line that is does not contain the recombinant DNA, known as a negative segregant.
As used herein an “enhanced trait” means a characteristic of a transgenic plant that includes, but is not limited to, an enhance agronomic trait characterized by enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In more specific aspects of this invention 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 an important aspect of the invention the enhanced trait is enhanced yield 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 intestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. “Yield” can be affected by many properties 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. Yield can also be affected by 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.
Increased yield of a transgenic plant of the present invention can be 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, kilo per hectare. For example, maize yield may be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, for example at 15.5 percent moisture. Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Recombinant DNA used in this invention can also be used to provide 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. Also of interest is the generation of transgenic plants that demonstrate enhanced yield with respect to a seed component that may or may not correspond to an increase in overall plant yield. Such properties include enhancements in seed oil, seed molecules such as tocopherol, protein and starch, or oil particular oil components as may be manifest by an alteration in the ratios of seed components.
A subset of the nucleic molecules of this invention includes fragments of the disclosed recombinant DNA consisting of oligonucleotides of at least 15, preferably at least 16 or 17, more preferably at least 18 or 19, and even more preferably at least 20 or more, consecutive nucleotides. Such oligonucleotides are fragments of the larger molecules having a sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO: 614, and find use, for example as probes and primers for detection of the polynucleotides of the present invention.
DNA constructs are assembled using methods well known to persons of ordinary skill in the art and typically comprise a promoter operably linked to DNA, the expression of which provides the enhanced agronomic trait. Other construct components may include additional regulatory elements, such as 5′ leaders and introns for enhancing transcription, 3′ untranslated regions (such as polyadenylation signals and sites), DNA for transit or signal peptides.
Numerous promoters that are functional 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 from the cauliflower mosaic virus. 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. 5,641,876, which discloses a rice actin promoter, 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. 09/757,089, which discloses a maize chloroplast aldolase promoter, U.S. patent application Ser. No. 08/706,946, which discloses a rice glutelin promoter, U.S. patent application Ser. No. 09/757,089, which discloses a maize aldolase (FDA) promoter, and U.S. Patent Application Ser. No. 60/310,370, which discloses a maize nicotianamine synthase promoter, 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.
In other aspects of the invention, preferential expression in plant green tissues is desired. Promoters for such use include those from genes such as Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit (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).
A promoter may be altered to contain multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with a promoter, expression 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 upstream (5′) or downstream (3′) to the coding sequence. In some instances, these 5′ enhancing elements are introns. Particularly useful as enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No. 5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase gene intron, the maize heat shock protein 70 gene intron (U.S. Pat. No. 5,593,874) and the maize shrunken 1 gene.
In other aspects of the invention, sufficient expression in plant seed tissues is desired to affect 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 supra), and peroxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol Biol. 31(6):1205-1216).
Recombinant DNA constructs prepared in accordance with the invention will also generally include a 3′ element that typically contains a polyadenylation signal and site. Well-known 3′ elements include those from Agrobacterium tumefaciens genes such as nos 3′, tml 3, tmr 3′, tms 3′, ocs 3′, tr7 3′, for example disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference; 3′ elements from plant genes such as wheat (Triticum aesevitum) heat shock protein 17 (Hsp17 3′), a wheat ubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene, a rice lactate dehydrogenase gene and a rice beta-tubulin gene, all of which are disclosed in U.S. published patent application 2002/0192813 A1, incorporated herein by reference; and the pea (Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3′), and 3′ elements from the genes within the host plant.
Constructs and vectors may also include a transit peptide for targeting of a gene 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.
Transgenic plants comprising or derived from plant cells of this invention transformed with recombinant DNA can be further enhanced by incorporating DNA providing other traits, e.g. herbicide and/or pest resistance traits. DNA for insect resistance can be derived from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. DNA for herbicide resistance can provide resistance to glyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil or norflurazon herbicides. For example, DNA encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S. Pat. Nos. 5,094,945; 5,627,061; 5,633,435 and 6,040,497 can impart glyphosate tolerance; DNA encoding a glyphosate oxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyl transferase (GAT) disclosed in U.S. Patent Application publication 2003/0083480 A1 can also impart glyphosate tolerance; DNA encoding a dicamba monooxygenase disclosed in U.S. Patent Application publication 2003/0135879 A1 can impart dicamba tolerance; DNA encoding bromoxynil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 can impart bromoxynil tolerance; DNA encoding phytoene desaturase (crtI) described in Misawa et al, (1993) Plant J. 4:833-840 can impart norflurazon tolerance; DNA encoding acetohydroxyacid synthase described in Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 can impart sulfonylurea herbicide tolerance; DNA encoding a BAR protein as disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 can impart glufosinate and bialaphos tolerance; DNA disclosed in U.S. Patent Application Publication 2003/010609 A1 can impart N-amino methyl phosphonic acid tolerance; DNA disclosed in U.S. Pat. No. 6,107,549 can impart pyridine herbicide resistance; DNA molecules and methods for imparting tolerance to multiple herbicides such as glyphosatc, atrazine, ALS inhibitors, isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat. No. 6,376,754 and U.S. Patent Application Publication 2002/0112260, all of said U.S. Patents and Patent Application Publications are incorporated herein by reference. Molecules and methods for imparting insect/nematode/virus resistance are disclosed in U.S. Pat. Nos. 5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent Application Publication 2003/0150017 A1, all of which are incorporated herein by reference.
Numerous methods for transforming plant cells with recombinant DNA are known in the art and may be used to make the transgenic plants, cells and nuclei of this invention. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. No. 5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn); U.S. Pat. No. 6,153,812 (wheat) and U.S. Pat. No. 6,365,807 (rice) and Agrobacterium-mediated transformation is described in U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,463,174 (canola); U.S. Pat. No. 5,591,616 (corn); U.S. Pat. No. 6,384,301 (soybean), U.S. Pat. No. 7,026,528 (wheat) and 6329571 (rice), all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation systems, 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 useful to introduce recombinant 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 recombinant DNA insertion in order to achieve site-specific integration, for example, to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function in plants including cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.
Transformation methods of this invention 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, hypocotyls, calli, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a tertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, hypocotyls, 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, for example various media and recipient target cells, transformation of immature embryo cells and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein by reference.
The seeds of transgenic plants can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plants line for selection of plants having an enhanced trait. In addition to direct transformation of a plant with a recombinant DNA, transgenic plants can be prepared by crossing a first plant having a recombinant DNA with a second plant lacking the DNA. For example, recombinant DNA can be introduced into a first plant line that is amenable to transformation to produce a transgenic plant which can be crossed with a second plant line to introgress the recombinant DNA into the second plant line. A transgenic plant with recombinant DNA providing an enhanced trait, e.g. enhanced yield, can be crossed with transgenic plant line having other recombinant DNA that confers another trait, for example herbicide resistance or pest resistance, to produce progeny plants having recombinant DNA that confers both traits. Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross will segregate such that some of the plants will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA, e.g. marker identification by analysis for recombinant DNA or, in the case where a selectable marker is linked to the recombinant, by application of the selecting agent such as a herbicide for use with a herbicide tolerance marker, or by selection for the enhanced trait. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line
In the practice of transformation DNA is typically introduced into chromosomes in the nuclei of only a small percentage of target plant cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or an herbicide. Any of the herbicides to which plants of this invention may be resistant are useful agents for selective markers. Plant cells containing potentially transformed nuclei are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aph IV), spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Examples of such selectable markers are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Selectable markers which provide an ability to visually identify transformants can also be employed, for example, 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.
Plant cells that survive exposure to the selective agent, or plant cells that have been scored positive in a screening assay, may be cultured in regeneration media and allowed to mature into plants having the transformed nuclei. Developing plantlets regenerated from transformed plant cells can be transferred to plant growth mix, and hardened off, for example, 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 regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue, and the plant species. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced, for example self-pollination is commonly used with transgenic corn. The regenerated transformed plant or its progeny seed or plants can be tested for expression of the recombinant DNA and selected for the presence of enhanced agronomic trait.
Transgenic plants derived from the plant cells of this invention are grown to generate transgenic plants having an enhanced trait as compared to a control plant and produce transgenic seed and haploid pollen of this invention. Such plants with enhanced traits are identified by selection of transformed plants or progeny seed for the enhanced trait. For efficiency a selection method is designed to evaluate multiple transgenic plants (events) comprising the recombinant DNA, for example multiple plants from 2 to 20 or more transgenic events. Transgenic plants grown from transgenic seed provided herein demonstrate improved agronomic traits that contribute to increased yield or other trait that provides increased plant value, including, for example, improved seed quality. Of particular interest are plants having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
Table 2 provides a list of proteins SEQ ID NO: 615 through SEQ ID NO: 1228 encoding DNA (“genes”) that are useful as recombinant DNA for production of transgenic plants with enhanced agronomic trait.
Column headings in Table 2 refer to the following information: “PEP SEQ ID” refers to a particular number of amino acid sequence in the Sequence Listing. “PHE ID” refers to an arbitrary number used to identify a particular recombinant polynucleotide corresponding to the translated protein encoded by the polynucleotide. “NUC SEQ ID” refers to a particular number of a nucleic acid sequence in the Sequence Listing which defines a polynucleotide used in a recombinant polynucleotide of this invention. “GENE NAME” refers to a common name for the recombinant polynucleotide. “GENE EFFECT” refers to the effect of the expressed polypeptide in providing yield improvement or other enhanced property. “CODING SEQUENCE” refers to peptide coding segments of the polynucleotide. “SPECIES” refers to the organism from which the polynucleotide DNA was derived.
Arabidopsis AtHAP3a
Arabidopsis thaliana
Zea mays
Zea mays
Zea mays
Arabidopsis CCA1
Arabidopsis thaliana
Zea mays
Arabidopsis thaliana
E. coli glnB
Escherichia coli
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Zea mays
Zea mays
Zea mays
Arabidopsis thaliana
Glycine max
Glycine max
Glycine max
Zea mays
Saccharomyces
cerevisiae
Glycine max
Zea mays
Zea mays
Zea mays
Anabaena SPP
Nostoc PCC7120
Zea mays
Zea mays
Glycine max
Glycine max
Glycine max
Zea mays
Zea mays
Saccharomyces
cerevisiae
Zea mays
Glycine max
Synechocystis
Synechocystis sp.
Zea mays
Saccharomyces
cerevisiae
Synechocystis S-
Synechocystis sp.
Glycine max
Aspergillus yA
Emericella nidulans
Synechocystis Sucrose
Synechocystis sp.
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Zea mays
Zea mays
Zea mays
Zea mays
Agrobacterium aiiA-
Agrobacterium
tumefaciens
Xylella aiiA-like
Xylella fastidiosa
Xanthomonas aiiA -
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Glycine max
Glycine max
Zea mays
Glycine max
Zea mays
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Xylella SAG13-like -
Xylella fastidiosa
Zea mays
Glycine max
Glycine max
Nostoc punctiforme
Nostoc punctiforme
Zea mays
Zea mays
Zea mays
Zea mays
Oryza sativa
Oryza sativa
Zea mays
Triticum aestivum
Zea mays
Zea mays
Oryza sativa
Oryza sativa
Zea mays
Zea mays
Zea mays
Zea mays
Oryza sativa
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Oryza sativa
Zea mays
Gossypium hirsutum
Zea mays
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Zea mays
Zea mays
Zea mays
Xylella adenylate
Xylella fastidiosa
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Zea mays
Oryza sativa
Oryza sativa
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Oryza sativa
Zea mays
Zea mays
Zea mays
Oryza sativa
Zea mays
Oryza sativa
Oryza sativa
Zea mays
Zea mays
Agrobacterium
Agrobacterium
tumefaciens
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Saccharomyces
cerevisiae
Glycine max
Synechocystis Rieske
Synechocystis sp.
Arabidopsis AGL15
Arabidopsis thaliana
Saccharomyces
cerevisiae
Zea mays
Zea mays
Zea mays
Oryza sativa
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Zea mays
E. coli betT
Escherichia coli
Xenorhabdus BetT-like
Xenorhabdus sp.
Zea mays
Oryza sativa
Zea mays
Zea mays
Zea mays
Glycine max
Glycine max
Glycine max
Zea mays
Glycine max
Glycine max
Glycine max
Glycine max
Zea mays
Arabidopsis salt-
Arabidopsis thaliana
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Glycine max
Zea mays
Oryza sativa
Arabidopsis thaliana
Arabidopsis thaliana
Zea mays
Arabidopsis thaliana
Zea mays
Zea mays
Zea mays
Glycine max
Escherichia coli
Nostoc sp. PCC 7120
Nostoc PCC7120
Brassica P-II
Brassica napus
Arabidopsis eskimo 1
Arabidopsis thaliana
Glycine max
Synechocystis 1-
Synechocystis sp.
Agrobacterium 1-
Agrobacterium
tumefaciens
Zea mays
Agrobacterium 1-
Agrobacterium
tumefaciens
Xylella 1-
Xylella fastidiosa
Zea mays
Zea mays
E. coli yagD
Escherichia coli
Saccharomyces
cerevisiae
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Oryza sativa
Saccharomyces
cerevisiae
Zea mays
Saccharomyces
cerevisiae
Oryza sativa
Zea mays
Glycine max
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Arabidopsis agl8 -
Arabidopsis thaliana
Zea mays
Zea mays
Zea mays
Nitrosomonas
Nitrosomonas
europaea dual function
europaea
Oryza sativa
Oryza sativa
Oryza sativa
Nostoc sp. PCC 7120
Nostoc PCC7120
Nostoc punctiforme
Nostoc punctiforme
Anabaena SPS C154
Nostoc PCC7120
Anabaena SPS C287
Nostoc PCC7120
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Saccharomyces
cerevisiae
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Arabidopsis thaliana
Arabidopsis SUT2
Arabidopsis thaliana
Arabidopsis SUT4
Arabidopsis thaliana
Oryza sativa
Oryza sativa
Aspergillis
Emericella nidulans
Zea mays
Zea mays
Glycine max
Saccharomyces
cerevisiae
Oryza sativa
Oryza sativa
Arabidopsis thaliana
Arabidopsis thaliana
Glycine max
Zea mays
Arabidopsis thaliana
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Glycine max
Glycine max
Oryza sativa
sorghum 14-3-3 10
Sorghum bicolor
sorghum 14-3-3 10 N-
Sorghum bicolor
Oryza sativa
Oryza sativa
Zea mays
Zea mays
Glycine max
Oryza sativa
Oryza sativa
Glycine max
Glycine max
Triticum aestivum
Triticum aestivum
Zea mays
Zea mays
Oryza sativa
Saccharomyces
cerevisiae
Oryza sativa
Glycine max
Oryza sativa
Saccharomyces
cerevisiae
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Saccharomyces
cerevisiae
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Oryza sativa
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Oryza sativa
Oryza sativa
Oryza sativa
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Zea mays
Zea mays
Glycine max
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Glycine max
Zea mays
Zea mays
Oryza sativa
Oryza sativa
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Glycine max
Glycine max
Glycine max
Zea mays
Zea mays
Zea mays
Arabidopsis LFY
Arabidopsis thaliana
Zea mays
Glycine max
Oryza sativa
Glycine max
Zea mays
Oryza sativa
Arabidopsis sucrose
Arabidopsis thaliana
Nostoc sp. PCC 7120
Nostoc PCC7120
Synechocystis sp. PCC
Synechocystis sp.
Nostoc punctiforme
Nostoc punctiforme
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Oryza sativa
Oryza sativa
Zea mays
Zea mays
Oryza sativa
Oryza sativa
Saccharomyces
cerevisiae
E. coli guaB -
Escherichia coli
Agrobacterium GuaB -
Agrobacterium
tumefaciens
Zea mays
Zea mays
Zea mays
Saccharomyces
cerevisiae
Arabidopsis Suc5 -
Arabidopsis thaliana
Glycine max
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Arabidopsis G748
Arabidopsis thaliana
Arabidopsis NAM (no
Arabidopsis thaliana
Glycine max
Zea mays
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Zea mays
Oryza sativa
Zea mays
Zea mays
Glycine max
Oryza sativa
Zea mays
Glycine max
Zea mays
Saccharomyces
cerevisiae
E. coli adhC -
Escherichia coli
Nostoc sp. PCC 7120
Nostoc PCC7120
Oryza sativa
Zea mays
Zea mays
Zea mays
Glycine max
Oryza sativa
Oryza sativa
Zea mays
Oryza sativa
Glycine max
Oryza sativa
Saccharomyces
cerevisiae
Zea mays
Glycine max
sorghum TTG1-like
Sorghum bicolor
Zea mays
Zea mays
Arabidopsis CRE1b
Arabidopsis thaliana
Arabidopsis HK2
Arabidopsis thaliana
Arabidopsis HK3
Arabidopsis thaliana
Zea mays
Oryza sativa
Oryza sativa
Oryza sativa
Arabidopsis nitrate
Arabidopsis thaliana
Zea mays
Zea mays
Oryza sativa
Oryza sativa
Arabidopsis thaliana
Synechocystis ssr3189 -
Synechocystis sp.
Synechocystis ssr2315 -
Synechocystis sp.
Triticum aestivum
Zea mays
Saccharomyces
cerevisiae
Arabidopsis thaliana
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Selection Methods for Transgenic Plants with Enhanced Agronomic Trait
Within a population of transgenic plants regenerated from plant cells transformed with the recombinant DNA in their nucleus many plants that survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Selection from the population is necessary to identify one or more transgenic plant cells that can provide plants with the enhanced trait. Transgenic plants having enhanced traits are selected from populations of plants regenerated or derived from plant cells transformed as described herein by evaluating the plants in a variety of assays to detect an enhanced trait, e.g. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. These assays also may take many forms including, but not limited to, direct screening for the trait in a greenhouse or field trial or by screening for a surrogate trait. Such analyses can be directed to detecting changes in the chemical composition, biomass, physiological properties, morphology of the plant. Changes in chemical compositions such as nutritional composition of grain can be detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in biomass characteristics can be made on greenhouse or field grown plants and can include plant height, stem diameter, root and shoot dry weights; and, for corn plants, ear length and diameter. Changes in physiological properties can be identified by evaluating responses to stress conditions, for example assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology can be measured by visual observation of tendency of a transformed plant with an enhanced agronomic trait to also appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other selection properties include 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, phenotypic characteristics of harvested grain may be evaluated, 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.
Although the transgenic plant nuclei, plant cells, plant pollen, plants and methods of this invention can be applied to a wide variety of plant species such as fruits, vegetables, grasses and trees, they are particularly useful when applied to crops such as corn, soybean, cotton, oilseed rape (canola), alfalfa, rice, wheat, sugar beet, sugar cane and sunflower.
The following examples illustrate aspects of the inventions.
This example illustrates the construction of a several alternative base transformation vectors for transferring recombinant DNA into the nucleus of a plant cell which can be regenerated into a transgenic plant of this invention.
A base transformation vector for bombardment transformation is produced using GATEWAY™ Destination (Invitrogen Life Technologies, Carlsbad, Calif.) vectors. pMON65154 is constructed for use in preparation of constructs comprising recombinant polynucleotides for corn transformation. The elements of the expression vector are summarized in Table 3 below. Generally, pMON65154 comprises a selectable marker expression cassette comprising a Cauliflower Mosaic Virus 35S promoter operably linked to a gene encoding neomycin phosphotransferase II (nptII). The 3′ region of the selectable marker expression cassette comprises the 3′ region of the Agrobacterium tumefaciens nopaline synthase gene (nos) followed 3′ by the 3′ region of the potato proteinase inhibitor II (pinII) gene. The plasmid pMON 65154 further comprises a plant expression cassette into which a gene of interest may be inserted using GATEWAY™ cloning methods. The GATEWAY™ cloning cassette is flanked 5′ by a rice actin 1 promoter, exon and intron and flanked 3′ by the 3′ region of the potato pinII gene. Using GATEWAY™ methods, the cloning cassette may be replaced with a gene of interest. The vector pMON65154, and derivatives thereof comprising a gene of interest, are particularly useful in methods of plant transformation via direct DNA delivery, such as microprojectile bombardment.
A similar plasmid vector, pMON72472, is constructed for use in Agrobacterium-mediated methods of plant transformation. pMON72472 comprises the gene of interest plant expression cassette, GATEWAY™ cloning, and plant selectable marker expression cassettes present in pMON65154. In addition, left and right T-DNA border sequences from Agrobacterium are added to the plasmid (Zambryski et al. (1982)). The right border sequence is located 5′ to the rice actin 1 promoter and the left border sequence is located 3′ to the pinII 3′ sequence situated 3′ to the nptlI gene. Furthermore, pMON72472 comprises a plasmid backbone to facilitate replication of the plasmid in both E. coli and Agrobacterium tumefaciens. The backbone has an oriV wide host range origin of DNA replication functional in Agrobacterium, a pBR322 origin of replication functional in E. coli, and a spectinomycin/streptomycin resistance gene for selection in both E. coli and Agrobacterium.
Vectors similar to those described above may be constructed for use in Agrobacterium or microprojectile bombardment maize transformation systems where the rice actin 1 promoter in the plant expression cassette portion is replaced with other desirable promoters including, but not limited to a corn globulin 1 promoter, a maize oleosin promoter, a glutelin 1 promoter, an aldolase promoter, a zein Z27 promoter, a pyruvate orthophosphate dikinase (PPDK) promoter, a soybean 7S alpha promoter, a peroxiredoxin antioxidant (Per1) promoter and a CaMV 35S promoter. Protein coding segments are amplified by PCR prior to insertion into vectors such as described above. Primers for PCR amplification can be 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. For GATEWAY cloning methods, PCR products are tailed with attB1 and attB2 sequences, purified then recombined into a destination vectors to produce an expression vector for use in transformation.
An alternative base transformation vector specifically useful for inserting a recombinant DNA construct into a chromosome in a nucleus in a corn plant cell by Agrobacterium-mediated transformation is pMON93039 which has the DNA in the nucleotide sequence of SEQ ID NO:27374 and the elements described in Table 4 and illustrated in
Agrobacterium
Arabidopsis EPSPS
tumefaciens Ti plasmid
Agrobacterium
E. coli
An alternative base transformation vector specifically useful for inserting a recombinant DNA construct into a chromosome in a nucleus in a dicot plant cell, e.g. soybean or oilseed rape, by Agrobacterium-mediated transformation is pMON82053 which has the DNA in the nucleotide sequence of SEQ ID NO:27375 and the elements described in Table 5 and illustrated in
Agrobacterium T-
tumefaciens Ti plasmid which functions to
Agrobacterium T-
E. coli plasmid ColE1.
An alternative base transformation vector specifically useful for inserting a recombinant DNA construct into a chromosome in a nucleus in a dicot plant cell, e.g. cotton, by Agrobacterium-mediated transformation is pMON99053 which has the DNA in the nucleotide sequence of SEQ ID NO:27376 and the elements described in Table 6 and illustrated in
Agrobacterium T-
petunia hsp70 5′ untranslated region
Agrobacterium tumefaciens Ti
Agrobacterium T-
E. coli
Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Each recombinant DNA coding for a protein identified in Table 2 is amplified by PCR prior to insertion into the insertion site within the gene of interest expression cassette of one of the base transformation vectors.
This example illustrates transformation methods useful in introducing recombinant DNA into corn chromosomes to produce the transgenic nuclei, plant cells, plants and pollen and the production and identification of transgenic corn plants and seed with an enhanced trait, i.e. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Plasmid vectors were prepared by cloning DNA identified in Table 1 and inserting the cloned DNA into a base transformation vector.
In Agrobacterium-mediated transformation corn embryo cells from a corn line that is readily transformable (e.g. corn line designated LH59) are grown in a greenhouse to produce ears that are harvested when the embryos are 1.5 to 2.0 mm in length. The ears are surface sterilized by spraying or soaking in 80% ethanol, followed by air drying. Immature embryos are isolated from individual kernels on surface sterilized ears. Prior to inoculation of maize cells, Agrobacterium cells are grown overnight at room temperature. Immature maize embryo cells are inoculated with Agrobacterium shortly after excision, and incubated at room temperature with Agrobacterium for 5-20 minutes. Immature embryo plant cells are then co-cultured with Agrobacterium for 1 to 3 days at 23° C. in the dark. Co-cultured embryos are transferred to selection media and cultured for approximately two weeks to allow embryogenic callus to develop. Embryogenic callus is transferred to culture medium containing 100 mg/L paromomycin and subcultured at about two week intervals. Transformed plant cells are recovered 6 to 8 weeks after initiation of selection.
For transformation by microprojectile bombardment maize immature embryos are isolated and cultured 3-4 days prior to bombardment. Prior to microprojectile bombardment, a suspension of gold particles is prepared onto which the desired recombinant DNA expression cassettes are precipitated. DNA is introduced into maize cells as described in U.S. Pat. Nos. 5,550,318 and 6,399,861 using the electric discharge particle acceleration gene delivery device. Following microprojectile bombardment, tissue is cultured in the dark at 27° C. Additional transformation methods and materials for making transgenic plants of this invention, for example, various media and recipient target cells, transformation of immature embryos and subsequence 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.
To regenerate transgenic corn plants a callus of transgenic plant cells resulting from transformation and selection is placed on media to initiate shoot development into plantlets which are transferred to potting soil for initial growth in a growth chamber at 26° C. followed by a mist bench before transplanting to 5 inch pots where plants are grown to maturity. The regenerated plants are self-fertilized and seed is harvested for use in one or more methods to select seeds, seedlings or progeny second generation transgenic plants (R2 plants) or hybrids, e.g. by selecting transgenic plants exhibiting an enhanced trait as compared to a control plant.
Transgenic corn plant cells are transformed with recombinant DNA from each of the genes identified in Table 1, e.g. with DNA having the nucleotide sequence of SEQ ID NO:1-614. Progeny transgenic 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 7.
This example illustrates transformation methods useful in introducing recombinant DNA into soybean chromosomes to produce the transgenic nuclei, plant cells, plants and pollen and the production and identification of transgenic soybean plants and seed with an enhanced trait, i.e. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Plasmid vectors were prepared by cloning DNA identified in Table 1 and inserting the cloned DNA into a base transformation vector.
For Agrobacterium-mediated transformation, soybean seeds are imbibed overnight and the meristem explants excised. The explants are placed in a wounding vessel. Soybean explants and induced Agrobacterium cells from a strain containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette are mixed no later than 14 hours from the time of initiation of seed imbibition, and wounded using sonication. Following wounding, explants are placed in co-culture for 2-5 days at which point they are transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots. Resistant shoots are harvested approximately 6-8 weeks and placed into selective rooting media for 2-3 weeks. Shoots producing roots are transferred to the greenhouse and potted in soil. Shoots that remain healthy on selection, but do not produce roots are transferred to non-selective rooting media for an additional two weeks. Roots from any shoots that produce roots off selection are tested for expression of the plant selectable marker before they are transferred to the greenhouse and potted in soil. Additionally, a DNA construct can be transferred into the genome of a soybean cell by particle bombardment and the cell regenerated into a fertile soybean plant as described in U.S. Pat. No. 5,015,580, herein incorporated by reference.
Transgenic soybean plant cells are transformed with recombinant DNA from each of the genes identified in Table 2, i.e. with DNA having the nucleotide sequence of SEQ ID NO:1-614. 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 7.
This example illustrates transformation methods useful in introducing recombinant DNA into cotton chromosomes to produce the transgenic nuclei, plant cells, plants and pollen and the production and identification of transgenic cotton plants and seed with an enhanced trait, i.e. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Plasmid vectors were prepared by cloning DNA identified min Table 1 and inserting the cloned DNA into a base transformation vector.
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: 614 are obtained by transforming with recombinant DNA from each of the genes identified in Table 2. 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.
This example illustrates transformation methods useful in introducing recombinant DNA into oilseed rape (canola) chromosomes to produce the transgenic nuclei, plant cells, plants and pollen and the production and identification of transgenic canola plants and seed with an enhanced trait, i.e. 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 2. Transgenic progeny plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil as reported in Example 7.
This example illustrates the identification of homologs of proteins encoded by the DNA identified in Table 2 which is used to provide transgenic seed and plants having enhanced agronomic traits. From the sequence of the homologs, homologous DNA sequence can be identified for preparing additional transgenic seeds and plants of this invention with enhanced agronomic traits.
An “All Protein Database” was 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 polynucleotide sequence provided herein was obtained, an “Organism Protein Database” was constructed of known protein sequences of the organism; it is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.
The All Protein Database was queried using amino acid sequences provided herein as SEQ ID NO: 615 through SEQ ID NO: 1228 using NCBI “blastp” program 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 was 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 of the polynucleotides provided herein, 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 was queried using polypeptide sequences provided herein as SEQ ID NO: 615 through SEQ ID NO: 1228 using NCBI “blastp” program with E-value cutoff of 1e-4. Up to 1000 top hits were kept. A BLAST searchable database was constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using NCBI “blastp” program with E-value cutoff of 1e-8. The hit with the best E-value was 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. Homologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 1229 through SEQ ID NO: 27373. These relationships of proteins of SEQ ID NO: 615 through 1228 and homologs of SEQ ID NO: 1229 through 27373 are identified in Table 7. The source organism for each homolog is found in the Sequence Listing.
This example illustrates identification of plant cells of the invention by screening derived plants and seeds for enhanced trait. Transgenic corn, soybean, cotton and canola seeds and plants with recombinant DNA from each of the genes identified in Table 2 are prepared using plant cells transformed with DNA that is stably integrated into a chromosome of the nuclei in a plant cell. Progeny transgenic 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 compared to control plants.
The physiological efficacy of transgenic plants, e.g. transgenic corn plants (tested as hybrids), can be tested for nitrogen use efficiency (NUE) traits in a high-throughput nitrogen (N) selection method compared to the measurements from testing of control plants.
Plants are allowed to grow for 28 days in a low nitrogen nutrient environment or for 23 days in a high nitrogen nutrient environment. The nitrogen nutrients are dispensed in the form of a macronutrient solution (see composition below) containing different amounts of nitrogen nutrient (2 mM NH4NO3 for a low nitrogen environment or 20 mM NIH4NO3 for a high nitrogen environment). Pots with corn plants a provided with 100 ml of nutrient solution three times a week on alternate days starting at eight days after planting for low nitrogen and ten days after planting for high nitrogen. Matting under the pots should be changed as needed to avoid nitrogen accumulation and buildup of root matter. Table 8 shows the amount of nutrients in the low and high nitrogen solutions.
After 28 days of plant growth under low nitrogen and 23 days of plant growth under high nitrogen, the following variables are measured: total shoot fresh mass (SFM) in grams (g), V6 leaf chlorophyll (LC) measured by Minolta SPAD meter in “relative units”, V6 leaf area (LA) measured in square centimeters (cm2), V6 leaf fresh mass (LFM) measured in grams and V6 leaf dry mass (LDM) measured in grams. Leaf fresh mass is measure on leaves that have been dried in a forced air oven at 80° C. for 3 days. From the collected data, two derived measurements are made: (1) Leaf chlorophyll area (LCA), which is a product of V6 relative chlorophyll content and its leaf area (relative units), indicates the spread of chlorophyll over the entire leaf area; and (2) specific leaf area (LSA), which is the ratio of V6 leaf area to its dry mass (cm2/g dry mass), serves as an indicator of nitrogen use efficiency (NUE).
A list of recombinant DNA constructs which improved growth in high nitrogen environments in transgenic plants is reported in Table 9.
A list of recombinant DNA constructs which improved growth in a low (limiting) nitrogen environment in transgenic plants is reported in Table 10.
Transgenic plants of this invention and control plants are planted in field without any supplemental nitrogen being applied. Nitrogen levels in the fields are analyzed in early April pre-planting, e.g. by collecting 30 sample soil cores from 0-24″ and 24 to 48″ soil layer and analyzing for nitrate-nitrogen, phosphorus(P), potassium(K), organic matter and pH. P, K and micronutrients are applied based upon soil test recommendations. Recombinant DNA constructs which improved growth without any nitrogen source in transgenic plants is reported in Table 11.
Many transgenic plants with recombinant DNA of this invention in a chromosome in the nucleus of their cells exhibit improved yield as compared to a control plant. Recombinant DNA constructs which show improved yield or enhancement in a surrogate indicators for yield in transgenic corn plants is reported in Table 12. Useful surrogate indicators for yield include source capacity (biomass), source output (sucrose and photosynthesis), sink components (kernel size, ear size, starch in the seed), development (light response, height, density tolerance), maturity, early flowering trait and physiological responses to high density planting, e.g., at 45,000 plants per acre.
Water use efficiency can be evaluated by high-throughput methods in greenhouse screening of potted corn plants. 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 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 degrees 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.
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 plants with recombinant DNA constructs which provide improved water use efficiency in transgenic corn plants are reported in Table 15.
Plants can be identified as having enhanced growth under cold stress by a cold germination assay using three sets of seeds. The first set consists of seeds that are F1 hybrids that are tested positive for the transgenic events and the recombinant DNA is expressed in the growing seed. The second set consists of control seeds, e.g. a nontransgenic, wild-type negative control made from the same genotype as the seeds in the first set. The third set consists 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), e.g. 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the assay.
Corn seeds are placed embryo side down in deionized water on blotter paper in a tray that is held at 9.7° C. for 24 days (no light) in a growth chamber. Germination counts are taken on days 10, 11, 12, 13, 14, 17, 19, 21, and 24. Seeds are considered germinated if the emerged radicle size is 1 cm. Tissue samples are collected at random on the last day of the experiment for confirmation of RNA expression. A germination index (GI) is calculated after the day 24 count using the formula:
GI=(Σ([T+1−n]*[Pi−Pi-1]))/T
where “T” is the number of days for the experiment, i.e. 24; “n” is the number of days after start on which a count is made; “P” is the percentage of seed germinated during a count; and “i” represents a particular count. Statistical differences are calculated between positive and wild type control.
Events of transgenic plants that showed a statistical significance at the p level of less than 0.05 relative to wild-type controls for improved seed growth under cold stress are reported in Table 16.
Cold stress tolerance for corn plants of this invention is also determined by a field trial under early spring planting around two weeks prior to the time local farmers plant corn to identify recombinant DNA constructs that confer enhanced cold vigor at germination and early seedling growth under cold stress. The same seeds also are planted under local optimal planting conditions such that the crop has little or no exposure to cold condition (normal treatment). Early planting cold field trials were carried out at five locations, Glyndon Minn., Mason Mich., Monmouth Ill., Dayton Iowa and Mystic Conn. At each location seeds are planted under both early and local optimal planting times with 3 repetitions of 20 kernels in a single row in a plot. Seeds are planted 1.5 to 2 inch deep into soil to avoid muddy conditions. Two temperature monitors are set up at each location to monitor both air and soil temperature daily. Seed emergence is defined as the time when the growing shoot breaks the soil surface. The number of emerged seedling in each plot is counted daily from the day the earliest plot begins to emerge until no significant changes in emergence occur. Seedling vigor is also rated on a scale of 1 to 9 at the V3-V4 stage before the average of corn plant height reaches 10 inches, where 1 represents excellent early growth, 5 represents average growth and 9 represents poor growth. Days to 50% emergence, maximum percent emergence and seedling vigor are calculated. Corn plants having recombinant DNA constructs showing enhanced cold vigor at germination and early seedling growth under the early spring planting field conditions are reported in Table 17.
E. Screens for Transgenic Plant Seeds with Increased Protein and/or Oil Levels
Transgenic plants with recombinant DNA producing seed with increased protein and/or oil content are determined by analyzing harvested seed. For example, near-infrared transmittance spectrometry is used to determine the composition of a bulk seed samples by analyzing for multiple traits in a single scan. Typical analysis parameters are provided in Table 18.
Transgenic plants with recombinant DNA constructs which improve seed compositions in terms of protein content are reported in Table 19.
Transgenic plants with recombinant DNA constructs which improve seed compositions in terms of oil content are reported in Table 20.
This example illustrates the identification of consensus amino acid sequences for the proteins encoded by recombinant DNA in transgenic seeds and plants disclosed herein and homologs.
ClustalW program was selected for multiple sequence alignments of the amino acid sequence of SEQ ID NO: 684, 704, 705, 706, 710, 719, 734, 735, 738, 743, 744, 745, 746, 761, 777, 779, 793, 804, 824, 891, 896, 900, 918, 924, 932, 957, 961, 1001, 1015, 1016, 1026, 1027, 1032, 1033, 1036, 1043, 1044, 1045, 1051, 1054, 1059, 1087, 1119, 1123, 1135, 1136, 1137, 1138, 1139, 1165, and their 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 amino acid sequence can be used to identify DNA corresponding to the full scope of this invention that is useful in providing transgenic plants, for example corn and soybean plants with enhanced agronomic traits, for example improved nitrogen use efficiency, improved yield, improved water use efficiency and/or improved growth under cold stress, due to the expression in the plants of DNA encoding a protein with amino acid sequence identical to the consensus amino acid sequence.
This example illustrates the identification by Pfam analysis of domain and domain module in proteins encoded by recombinant DNA in the transgenic plants and seeds disclosed herein. The amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam domain modules and individual protein domain for the proteins of SEQ ID NO: 615 through 1228 are shown in Table 21 and Table 22 respectively. The Hidden Markov model databases for the identified protein 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: 668 is characterized by three Pfam domains, i.e., AdoHcyase, 2-Hacid_dh_C and AdoHcyase_NAD. See also the protein with amino acids of SEQ ID NO: 659 which is characterized by five copies of the Pfam domain “Arm”. In Table 22 “score” is the gathering score for the Hidden Markov Model of the domain which exceeds the gathering cutoff reported in Table 23.
This example illustrates the preparation and identification by selection of transgenic seeds and plants derived from transgenic plant cells of this invention having recombinant DNA in a chromosome in the nucleus of such cells. The plants and seeds are identified by screening for a transgenic plant having an enhanced agronomic trait imparted by expression of a protein selected from the group including the homologous proteins identified in Example 6. Transgenic plant cells of corn, soybean, cotton, canola, wheat and rice are transformed with recombinant DNA for expressing each of the homologs identified in Example 6. Plants are regenerated from the transformed plant cells and used to produce progeny plants and seed that are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Plants are identified exhibiting enhanced traits imparted by expression of the homologous proteins.
This application is a continuation in part of application Ser. No. 10/678,588, filed Oct. 2, 2003, which claims the benefit under 35 U.S.C. 119(e) of provisional application Ser. No. 60/415,758, filed Oct. 2, 2002, 60/425,157, filed Nov. 8, 2002 and 60/463,787, filed Apr. 18, 2003, the disclosures of which are incorporated herein by reference in their entirety. This application is a continuation in part of application Ser. No. 10/679,063, filed Oct. 2, 2003, which is currently abandoned with a petition to revive and which claims the benefit under 35 U.S.C. 119(c) of provisional application 60/415,758, filed Oct. 2, 2002, the disclosures of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60415758 | Oct 2002 | US | |
| 60425157 | Nov 2002 | US | |
| 60463787 | Apr 2003 | US | |
| 60415758 | Oct 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13199773 | Sep 2011 | US |
| Child | 14998668 | US | |
| Parent | 11982680 | Nov 2007 | US |
| Child | 13199773 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10678588 | Oct 2003 | US |
| Child | 11982680 | US | |
| Parent | 10679063 | Oct 2003 | US |
| Child | 11982680 | US |
