CN101535483A

CN101535483A - Modulating plant nitrogen levels

Info

Publication number: CN101535483A
Application number: CNA2005800469973A
Authority: CN
Inventors: R·施内贝格尔; E·马戈勒斯-克拉克; J·派克; B·詹科沃斯基; S·C·鲍伯齐恩
Original assignee: Ceres Inc
Current assignee: Ceres Inc
Priority date: 2004-12-16
Filing date: 2005-12-02
Publication date: 2009-09-16

Abstract

Methods and materials for modulating (e.g., increasing or decreasing) nitrogen levels in plants are disclosed. For example, nucleic acids encoding nitrogen-modulating polypeptides are disclosed as well as methods for using such nucleic acids to transform plant cells. Plants and plant products having increased nitrogen levels are also disclosed.

Description

Regulate plant nitrogen levels

The cross reference of related application

According to 35U.S.C. § 119, the right of priority of the U.S. Provisional Application that the application requires to submit on December 16th, 2004 U.S. Provisional Application is submitted to number on August 2nd, 60/637,311 and 2005 number 60/705,119, it is for referencial use to include its full content in this paper.

Technical field

The application provides method and the material that relates to nitrogen level in adjusting (as improving or the reducing) plant.For example, the application provides plant and the plant of preparation nitrogen level rising and the material and the method for plant prod that nitrogen level raises.

Background of invention

Photoautotrophy production nitrogen-containing organic compound is most important to plant metabolism, g and D.In many farm crop kinds, the protein of the plant material of results and aminoacids content have very important rural economy to be worth.The nitrogen of optical drive (N) assimilation has been evolved to carrying out synchronously with photosynthesis and respiration and integrating in the leaf.Reducing the generation of carbon (C) and it in the photosynthesis reoxidizes in respiration for producing and inorganic N is mixed the required energy of amino acid and the C skeleton is essential.On the contrary, need N to assimilate to keep the output of organic C and N.Be accompanied by that photorespiration is metabolic carries out, it is complicated more that this network becomes.The coordination of N assimilation speed and C and N assimilation is under the multifactor control of many signals, and these signals provide the information of C and N state.Need under different nitrogen conditions, to improve the composition and the method for nitrogen content in the plant.

Summary of the invention

The application provides and has related to method and the material that nitrogen content is subjected to the plant of adjusting (as improving or reducing).For example, transgenic plant and vegetable cell that the application provides nitrogen level to raise are used to produce the transgenic plant of nitrogen level rising and the nucleic acid of vegetable cell, and the plant of preparation nitrogen level rising and the method for vegetable cell.Can cultivate this kind of plant and vegetable cell, to produce the seed that nitrogen content raises.These seeds can be used for producing grain and the animal-feed that nutrition (as protein) content raises, and this helps grain-production person and human consumer.Though be not subject to any concrete mode of action, nitrogen regulatory polypeptide provided herein may have the activity as translocator.For example, polypeptide provided herein can participate in the transhipment of nitrogen (as the organonitrogen of peptide or amino acid form or the inorganic nitrogen of ammonium or nitrate form).

In one embodiment, provide a kind of method of regulating nitrogen level in the plant.This method comprises isolating nucleic acid introduced plant cell, it is 80% or the nucleotide sequence of higher polypeptide that described nucleic acid comprises coding and the sequence homogeny of aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:4-18 and consensus sequence shown in Figure 1, nitrogen level in the plant tissue that this vegetable cell produces and the respective horizontal that does not contain in the control plant tissue of this nucleic acid are variant.

In another embodiment, provide a kind of method of regulating nitrogen level in the plant.This method comprises isolating nucleic acid introduced plant cell, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny of aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:16 and consensus sequence shown in Figure 1, nitrogen level in the plant tissue that this vegetable cell produces and the respective horizontal that does not contain in the control plant tissue of this nucleic acid are variant.

In another embodiment, provide a kind of method of regulating nitrogen level in the plant.This method comprises isolating nucleic acid introduced plant cell, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny of aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:16, nitrogen level in the plant tissue that this vegetable cell produces and the respective horizontal that does not contain in the control plant tissue of this nucleic acid are variant.

Described sequence homogeny can be 85% or higher, 90% or higher or 95% or higher.Described nucleotide sequence codified comprises the polypeptide corresponding to the aminoacid sequence of SEQ ID NO:2.Described nucleotide sequence codified comprises the polypeptide corresponding to the aminoacid sequence of SEQ ID NO:4.Described nucleotide sequence codified comprises the polypeptide corresponding to the aminoacid sequence of consensus sequence shown in Figure 1.Described difference can be that nitrogen level raises.

Described isolating nucleic acid operability is connected in regulatory region.This regulatory region can be the tissue specificity regulatory region.This tissue specificity regulatory region can be a promotor.Described promotor can be selected from: YP0092, PT0676, PT0708, the rapeseed protein promotor, the Arcelin-5 promotor, the Kidney bean protein gene promoter, the Trypsin inhibitor SBTI promotor, the ACP promotor, the stearyl-ACP desaturase gene, the soybean α 1 subunit promotor of beta-conglycinin, the oleosin promotor, 15kD zein promotor, 16kD zein promotor, 19kD zein promotor, 22kD zein promotor, 27kD zein promotor, the Osgt-1 promotor, beta-amylase gene promoter or hordein gene promotor.Described promotor can be selected from: PT0613, PT0672, PT0678, PT0688, PT0837, YP0128, YP0275, PT0625, PT0660, PT0683 or PT0758.Described regulatory region can be the promotor of wide expression.The promotor of wide expression can be selected from: p13879, p32449,21876, p326, YP0158, YP0214, YP0380, PT0848, PT0633, YP0050, YP0144 or YP0190.Described regulatory region can be an inducible promoter.

This plant can be a dicotyledons.This plant can be the member of Btassica (Brassica), Glycine (Glycine), Gossypium (Gossypium), Helianthus (Helianthus), Lactuca (Lactuca), tomato genus (Lycopersicon), Solanum (Solanum), Vitis (Vitis), Pisum (Pisum), Medicago (Medicago), safflower genus (Carthamus), Arachis (Arachis), Olea (Olea), linum (Linum) or Trifolium (Trifolium).This plant can be a monocotyledons.This plant can be the member of Zea (Zea), Triticum (Triticum), Hordeum (Hordeum), Secale (Secale), Oryza (Oryza), triticale genus (Triticosecale), Avena (Avena), Musa (Musa), oil palm genus (Elaeis), ladder forage spp (Phleum) or sorghum (Sorghum).Described tissue can be a seed tissue.

A kind of method of producing plant tissue also is provided.Described method comprises cultivates a kind of vegetable cell that contains isolating nucleic acid, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny of aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:4-18 and consensus sequence shown in Figure 1, nitrogen level in the described tissue and the respective horizontal that does not contain in the control plant tissue of this nucleic acid are variant.

In another embodiment, provide a kind of method of producing plant tissue.Described method comprises cultivates a kind of vegetable cell that contains isolating nucleic acid, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny of aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:16 and consensus sequence shown in Figure 1, nitrogen level in the described tissue and the respective horizontal that does not contain in the control plant tissue of this nucleic acid are variant.

In another embodiment, provide a kind of method of producing plant tissue.Described method comprises cultivates a kind of vegetable cell that contains isolating nucleic acid, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny of aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:5 and SEQ ID NO:16, nitrogen level in the described tissue and the respective horizontal that does not contain in the control plant tissue of this nucleic acid are variant.

This plant tissue can be the dicotyledons tissue.This plant tissue can be the member of Btassica, Glycine, Gossypium, Helianthus, Lactuca, tomato genus, Solanum, Vitis, Pisum, Medicago, safflower genus, Arachis, Olea, linum or Trifolium.This plant tissue can be the monocotyledons tissue.This plant tissue can be the member of Zea, Triticum, Hordeum, Secale, Oryza, triticale genus, Avena, Musa, oil palm genus, ladder forage spp or sorghum.Described tissue can be a seed tissue.

A kind of vegetable cell also is provided.Described vegetable cell comprises a kind of isolating nucleic acid, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny of aminoacid sequence that is selected from down group: SEQID NO:2, SEQ ID NO:4-18 and consensus sequence shown in Figure 1, nitrogen level in the plant tissue that this vegetable cell produces and the respective horizontal that does not contain in the control plant tissue of this nucleic acid are variant.

In another embodiment, provide a kind of vegetable cell.Described vegetable cell comprises a kind of isolating nucleic acid, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny of aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:16 and consensus sequence shown in Figure 1, nitrogen level in the plant tissue that this vegetable cell produces and the respective horizontal that does not contain in the control plant tissue of this nucleic acid are variant.

In another embodiment, provide a kind of vegetable cell.Described vegetable cell comprises a kind of isolating nucleic acid, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny of aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:16, nitrogen level in the plant tissue that this vegetable cell produces and the respective horizontal that does not contain in the control plant tissue of this nucleic acid are variant.

This plant can be a dicotyledons.This plant can be the member of Btassica, Glycine, Gossypium, Helianthus, Lactuca, tomato genus, Solanum, Vitis, Pisum, Medicago, safflower genus, Arachis, Olea, linum or Trifolium.This plant can be a monocotyledons.This plant can be the member of Zea, Triticum, Hordeum, Secale, Oryza, triticale genus, Avena, Musa, oil palm genus, ladder forage spp or sorghum.Described tissue can be a seed tissue.

A kind of transgenic plant also are provided.Described transgenic plant comprise any above-mentioned vegetable cell.The filial generation of described transgenic plant also is provided.The nitrogen level of described filial generation is variant with the nitrogen level in the corresponding control plant that does not contain isolating nucleic acid.Seed and the nutritive issue of transgenic plant also are provided.The grain and the feed of the nutritive issue that comprises transgenic plant are provided in addition.

Except as otherwise noted, all scientific and technical terminologies used herein are identical with the implication of one skilled in the art's common sense of the present invention.Though method and the material similar or equivalent to content described herein can be used for implementing the present invention, and suitable method and material are described below.All that this paper is mentioned are delivered thing, patent application, patent and clear soup reference, and to include this paper in full in for referencial use.Having under the situation of conflict, be as the criterion with this specification sheets (comprising definition).In addition, material, method and embodiment only are illustrative, can not limit the present invention.

The details of one or more embodiments of the present invention have been set forth in accompanying drawing and the following specification sheets.From this specification sheets and accompanying drawing and claims, can understand other features, objects and advantages of the present invention.

Description of drawings

Fig. 1 is SEQ ID NO:4 and straight comparison to homologous amino acid sequence SEQ ID NO:5-7, SEQ ID NO:9-12, SEQ ID NO:14 and SEQ ID NO:16-18.Listed the consensus sequence of determining by comparison.

Detailed Description Of The Invention

The invention describes method and the material of the plant, plant product, plant tissue and the plant cell that relate to nitrogen level modulated (as improving or reducing). For example, plant and plant cell that the application provides nitrogen level to raise, and the method for producing this Plants and plant cell. Described method can comprise the nucleic acid transformed plant cell with coding nitrogen regulatory polypeptide, and the expression of wherein said polypeptide causes nitrogen level to raise. Can cultivate the plant and the plant cell that produce in this way, the seed that raises to produce nitrogen level. These seeds can be used for producing grain and the animal feed that nutrients (such as protein) content raises, and this is conducive to grain-production person and consumer.

Polypeptide

Term used herein " polypeptide " refers to the compound of two or more subunit amino acid, amino acid analogue or other peptide mimics, no matter posttranslational modification (such as phosphorylation or glycosylation). Subunit can pass through peptide bond or other key, connects such as ester bond or ehter bond. Term " amino acid " refers to natural and/or non-natural or synthetic amino acid, comprises the D/L optical isomer. This definition comprises full length protein and analog, mutant and fragment.

Described herein is the nitrogen regulatory polypeptide. When expressing, the nitrogen regulatory polypeptide can effectively regulate nitrogen level in plant or plant cell. The adjusting of nitrogen level can be to improve or reduce nitrogen level with respect to the respective horizontal in the check plant. The nitrogen regulatory polypeptide can be the transhipment polypeptide, such as oligopeptides transhipment polypeptide.

The nitrogen regulatory polypeptide can be proton dependence oligopeptides transhipment (POT) family polypeptides. It is reported that the POT family polypeptides has participated in being accompanied by the proton picked-up and taken in the process of little peptide. SEQ ID NO:2 and SEQ ID NO:4 list the arabidopsis of knowing clearly (Arabidopsis) clone's amino acid sequence, this paper is called Ceres cDNA ID 2998984 (SEQ ID NO:1) and Ceres clone 117581 (SEQ ID NO:3), has separately the characteristic PTR2 domain of peptide transhipment polypeptide.

The nitrogen regulatory polypeptide can comprise SEQ ID NO:2 or the listed amino acid sequence of SEQ ID NO:4. Perhaps, the nitrogen regulatory polypeptide can be homologue, straight homologues or the variant with polypeptide of SEQ ID NO:2 or the listed amino acid sequence of SEQ ID NO:4. For example, the nitrogen regulatory polypeptide can have the monoamino-acid sequence, the sequence homogeny of this sequence and SEQ ID NO:2 or the listed amino acid sequence of SEQ ID NO:4 is at least 40%, such as 41%, 45%, 47%, 48%, 49%, 50%, 51%, 52%, 56%, 57%, 60%, 61%, 62%, 63%, 64%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%. In some embodiments, the nitrogen regulatory polypeptide comprises with SEQ ID NO:2 sequence homogeny and is at least 40% amino acid sequence, and the N end of this polypeptide comprises chloroplast targeted burst.

Fig. 1 provides the amino acid sequence of the polypeptide straight homologues with the listed amino acid sequence of SEQ ID NO:4, and consensus sequence. Comparison such as the amino acid sequence about SEQ ID NO:4, and is determined that everybody is set up modal amino acid or amino acid type, thereby is determined the consensus amino acid sequences of this straight homologues from the amino acid sequence of various species. For example, the comparison of Fig. 1 provides following amino acid sequence Ceres clone 117581 (SEQ ID NO:4), Ceres clone: 328378 (SEQ ID NO:5), gi|2655098 (SEQ ID NO:6), gi|34895718 (SEQ ID NO:7), gi|4102839 (SEQ ID NO:9), gi|31088360 (SEQ ID NO:10), gi|6635838 (SEQ ID NO:11), gi|56784523 (SEQ ID NO:12), gi|50059161 (SEQ ID NO:14), Ceres clone: 352232 (SEQ ID NO:16), gi|33411520 (SEQ ID NO:17) and gi|31429847 (SEQ ID NO:18). Other straight homologues comprises gi|50933627 (SEQ ID NO:8), gi|56784524 (SEQ ID NO:13) and gi|6409176 (SEQ ID NO:15).

In some cases, the nitrogen regulatory polypeptide can comprise the polypeptide that is at least 80% (identical such as 80%, 85%, 90%, 93%, 95%, 97%, 98% or 99% sequence) with the sequence homogeny corresponding to the amino acid sequence of consensus sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ DD NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or Fig. 1.

Should be understood that many nucleic acid codifieds have the polypeptide of specific amino acid sequence. The degeneracy of genetic code is well known in the art; Namely for many amino acid, more than one nucleotide triplet is arranged as a kind of amino acid whose codon. For example, suitable codon bias table is modified the codon in the coded sequence of given nitrogen regulatory polypeptide in the available concrete plant species, to obtain the optimal expression in this plant species.

The nitrogen regulatory polypeptide of recombinant nucleic acid coding can be the natural N regulatory polypeptide, i.e. one or more additional copies of naturally occurring nitrogen regulatory polypeptide coded sequence in the cell. Perhaps, the nitrogen regulatory polypeptide may with this cell allos, as, the transgene tomato platymiscium can contain the coded sequence from the transhipment polypeptide of soybean plants.

The nitrogen regulatory polypeptide can comprise the additional amino acid that does not participate in the nitrogen adjusting, therefore may be longer than the polypeptide that does not comprise additional amino acid. For example, the nitrogen regulatory polypeptide can comprise the amino acid sequence as reporter. This nitrogen regulatory polypeptide can be that green fluorescent protein (GFP) polypeptide and SEQ ID NO:2 merge fusion that form or yellow fluorescence protein (YFP) polypeptide and SEQ ID NO:4 fusion formation. In some embodiments, the nitrogen regulatory polypeptide comprises purification tag or the targeting sequencing that is added in amino or carboxyl terminal.

Can identify by nucleotides and peptide sequence compare of analysis and be applicable to nitrogen regulatory polypeptide material standed for of the present invention. For example, nucleotides or peptide sequence database are inquired about the straight homologues that can identify the nitrogen regulatory polypeptide. Sequence analysis can comprise with known nitrogen regulatory polypeptide amino acid sequence carries out BLAST, mutual BLAST or PSI-BLAST analysis to the Non-redundant data storehouse. The sequence homogeny can be accredited as material standed for greater than 40% protein in the database, with its applicability as the nitrogen regulatory polypeptide of further evaluation. If necessary, can check by hand these material standed fors, need the further quantity of the material standed for of evaluation to reduce. Carry out manual inspection by selecting as if to have the material standed for of suspecting the domain (such as conservative functional domain) that is present in the nitrogen regulatory polypeptide.

Identify that in template or theme polypeptide conserved region can be conducive to produce the variant of wild type nitrogen regulatory polypeptide. Can identify by the following method conserved region: be repetitive sequence in the one-level amino acid sequence of locating template polypeptide, form some secondary structure (such as spiral and β-pleated sheet), set up the zone of positively charged or electronegative domain or representative albumen motif or domain. Referring to for example, the consensus sequence of various albumen motifs and domain has been described in the Pfam website such as WWW sanger.ac.uk/Pfam/ and genome.wustl.edu/Pfam. Sonnhammer etc., 1998, Nucl.Acids Res.26:320-322; Sonnhammer etc., 1997, Proteins 28:405-420; With Bateman etc., 1999, Nucl.Acids Res.27:260-262 has described the explanation of the contained information of Pfam database.

Also can determine conserved region by the be closely related sequence of identical or related polypeptide of species of comparison. The species that are closely related are preferably the species of same section. In some embodiments, the sequence of two different plant species of comparison is just enough. For example, the sequence from arabidopsis (Arabidopsis) and maize (Zea mays) can be used for identifying one or more conserved region.

Usually, the amino acid sequence homogeny is at least about 40% polypeptide and can be used for identifying conserved region. The amino acid sequence homogeny of the conserved region of related polypeptide is at least 45% (being at least 50%, at least 60%, at least 70%, at least 80% or at least 90% such as the amino acid sequence homogeny). In some embodiments, the amino acid sequence homogeny of the conserved region of target polypeptide and template polypeptide is at least 92%, 94%, 96%, 98% or 99%. Can derive the amino acid sequence homogeny from amino acid or nucleotide sequence. In some cases, can identify the domain of high conservative in the nitrogen regulatory polypeptide. The nitrogen regulatory polypeptide that these conserved region can be used for identifying similar on the function (directly to homology).

In some cases, can be according to the synthetic suitable nitrogen regulatory polypeptide of total functional domain and/or conserved region in the homology nitrogen regulatory polypeptide. Domain is the basic contiguous amino acid group that can be used in the polypeptide of profiling protein matter family and/or protein part. This domain has " fingerprint " or " signature ", and " fingerprint " or " signature " can comprise conservative (1) primary sequence, (2) secondary structure and/or (3) three-dimensional conformation. Usually, each domain is with specific external and/or activity in vivo is relevant. The length of domain can be 10 amino acid-400 amino acid, such as 10-50 amino acid, or 25-100 amino acid, or 35-65 amino acid, or 35-55 amino acid, or 45-60 amino acid, or 200-300 amino acid, or 300-400 amino acid.

Can be by above-mentioned homology peptide sequence Analysis and Identification apokoinou construction territory and conserved region. Can be by the applicability of research evaluation polypeptide as the nitrogen regulatory polypeptide that have complementary functions.

Nucleic acid

This paper provides isolating nucleic acid. In this article, term " nucleic acid " and " polynucleotides " are used interchangeably, and they refer to RNA and DNA, comprise cDNA, genomic DNA, synthetic DNA and contain the DNA (or RNA) of nucleic acid analog. Polynucleotides can have any three-dimensional structure. Nucleic acid can be two strands or strand (being positive-sense strand or antisense strand). The non-limitative example of polynucleotides comprises the DNA isolation of gene, genetic fragment, extron, introne, mRNA (mRNA), transfer RNA, rRNA, siRNA, Microrna, ribozyme, cDNA, recombination of polynucleotide, branch's polynucleotides, plasmid, carrier, arbitrary sequence, isolation of RNA, nucleic acid probe and primer and the nucleic acid analog of arbitrary sequence.

Isolating nucleic acid can be the dna molecular of (for example) natural generation, if in natural generation genome usually closely side joint be removed or do not exist in one of nucleotide sequence of this dna molecular. Therefore, isolating nucleic acid includes but not limited to: the dna molecular that is independent of other sequence, exists with independent molecule (such as the nucleic acid of chemical synthesis, perhaps polymerase chain reaction (PCR) or restriction endonuclease are processed cDNA or the genomic DNA fragment that produces). Isolating nucleic acid also refers to mix DNA, autonomously replicating plasmid, the virus of carrier or mixes protokaryon or the dna molecular of Eukaryotic genomic DNA. In addition, isolating nucleic acid can comprise engineered nucleic acid, such as the dna molecular as hybridization or the part of integrative nucleic acid. Be present in (for example) cDNA library or genomic library, or contain hundreds of the nucleic acid to millions of other nucleic acid in the gel film of genomic DNA restrictive diges-tion thing, be not considered to isolating nucleic acid.

The available standards technology is produced isolated nucleic acid molecule. For example, available polymerase chain reaction (PCR) technology obtains to contain the isolating nucleic acid of nucleotide sequence described herein. PCR refers to method or the technology of amplifying target nucleic acid. Can adopt the PCR particular sequence that from DNA and RNA, increases, comprise the sequence in total genomic dna or the total cell RNA. For example, " PCR primer: laboratory manual " (PCR Primer:A Laboratory Manual), Dieffenbach and Dveksler compile, and Cold Spring Harbor Laboratory Press has described various PCR methods in 1995. Usually, with the sequence information design of area-of-interest end or farther place and the same or analogous Oligonucleolide primers of antisense strand sequence of template to be amplified. Also can obtain and the locus specificity nucleotide sequence can be modified the various PCR schemes of introducing template nucleic acid. The isolating nucleic acid of chemical synthesis can be mononucleotide molecule (synthesizing as carrying out automated DNA with the phosphoramidite technology in 3 '-5 ' direction) or a series of oligonucleotides. For example, can synthesize one or more pairs of long oligonucleotides of containing required sequence (as〉100 nucleotides), each is to containing complementary short-movie section (according to appointment 15 nucleotides), in order to form duplex during to annealing at oligonucleotides. Extend oligonucleotides with archaeal dna polymerase, every pair of oligonucleotides produces single, double chain nucleic acid molecules, then can connect into carrier. Also can obtain isolating nucleic acid of the present invention by the DNA of the natural generation of mutagenesis (for example).

Term used herein " sequence homogeny percentage " refers to the same degree between any given search sequence and the subject nucleotide sequence. The length of subject nucleotide sequence is generally greater than 80% of search sequence length, as greater than 82,85,87,89,90,93,95,97,99,100,105,110,115 or 120% of search sequence length. With computer program ClustalW (1.83 editions, default parameters) inquiry nucleic acid or amino acid sequence are compared with one or more theme nucleic acid or amino acid sequence, carry out the comparison (overall comparison) of nucleic acid or protein sequence in total length. Chenna etc. (2003) Nucleic Acids Res 31 (13): 3497-500.

ClustalW calculates the optimum matching between search sequence and one or more subject nucleotide sequences, and they measure homogeny, similarity and difference by comparison.The breach of one or more residues can be inserted in search sequence, subject nucleotide sequence or the two, at utmost to carry out sequence alignment.In the paired fast comparison of nucleotide sequence, adopt following default parameters: word length: 2; Window size: 4; Methods of marking: percentage ratio; Top diagonal lines quantity: 4; Breach point penalty: 5.The multiple ratio centering of nucleotide sequence, adopt following parameter: the open point penalty of breach: 10.0; Breach extends point penalty: 5.0; Weight conversion: be.In the paired fast comparison of protein sequence, adopt following parameter: word length: 1; Window size: 5; Methods of marking: percentage ratio; Top diagonal lines quantity: 5; Breach point penalty: 3.The multiple ratio centering of protein sequence, adopt following parameter: weighting matrix: piece and (blosum); The open point penalty of breach: 10.0; Breach extends point penalty: 0.05; Wetting ability breach: open; Wetting ability residue: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg and Lys; Residue specificity breach point penalty: open.Output is the sequence alignment that concerns between the reflection sequence.Can go up operation ClustalW in (for example) Beile drug research pioneer institute website (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) with in European bioinformation research institute Website (ebi.ac.uk/clustalw).In order to determine " the homogeny percentage ratio " between search sequence and the subject nucleotide sequence, the base of coupling or amino acid whose quantity multiply by 100 then divided by coupling and mismatched bases or amino acid whose sum in the usefulness comparison.It should be noted that the homogeny percent value can be rounded to behind the radix point one.For example, 78.11,78.12,78.13 and 78.14 are rounded to 78.1, and 78.15,78.16,78.17,78.18 and 78.19 are rounded to 78.2.Also it should be noted that length value integer always.

When mentioning nucleic acid, term " exogenous " refers to that this nucleic acid is the part of recombinant nucleic acid construction, perhaps is not under its natural surroundings.For example, exogenous nucleic acid can be the sequence of introducing another species from a kind of species, i.e. heterologous nucleic acids.Usually, by the recombinant nucleic acid construction this exogenous nucleic acid is introduced other species.Exogenous nucleic acid also can be the natural sequence that has and introduce this biological cell again of certain organism.Usually can be by being connected in the non-natural sequence of this exogenous nucleic acid, as the existence of the non-natural regulating and controlling sequence of native sequences in the side joint recombinant nucleic acid construction, the exogenous nucleic acid and the naturally occurring sequence that will comprise native sequences make a distinction.In addition, the exogenous nucleic acid of stable conversion generally is incorporated on the position in addition, position of finding native sequences.Should be understood that and exogenous nucleic acid can be introduced progenitor cell, but not the cell of being studied.For example, the transgenic plant that contain exogenous nucleic acid can be the filial generations of hybridizing between stable conversion plant and the non-transgenic plant.Think that this filial generation contains exogenous nucleic acid.

This paper also provides the recombination to construct thing, and available recombination to construct thing transforms plant or vegetable cell, to regulate nitrogen level.The recombinant nucleic acid construction comprises the nucleic acid of the nitrogen regulatory polypeptide described herein of encoding, and its operability is connected in and is applicable to the regulatory region of expressing this nitrogen regulatory polypeptide in plant or cell.Therefore, nucleic acid can comprise the nucleotide sequence of the listed any nitrogen regulatory polypeptide of coding SEQ IDNO:2, SEQ ID NO:4-18 and consensus sequence shown in Figure 1.

The carrier that contains nucleic acid as described herein also is provided." carrier " is replicon, as plasmid, phage or clay, wherein may insert another DNA sections, so that duplicate the sections of insertion.Usually, carrier can duplicate when suitable controlling elements links to each other.The suitable carriers skeleton for example comprises: this area common carrier, and as plasmid, virus, artificial chromosome, BAC, YAC or PAC.Term " carrier " comprises clone and expression vector, and virus vector and integrative vector." expression vector " is the carrier that comprises control region.Suitable expression vector includes but not limited to: plasmid and derived from (for example) phage, baculovirus and retroviral virus vector.Many carriers and expression system can available from such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA) and Invitrogen/LifeTechnologies (Carlsbad, company such as CA).

Carrier provided herein also for example can comprise, replication orgin, scaffold attached region (SAR) and/or mark.Marker gene can make vegetable cell produce the selectivity phenotype.For example, mark can produce the biocides resistance, as microbiotic (as kantlex, G418, bleomycin or Totomycin) resistance, or weedicide (as chlorine sulphur dragon (chlorosulfuron) or glufosinates) resistance.In addition, expression vector can comprise the sequence label that helps the operation or the detection (as purifying or location) of express polypeptide through design.Sequence label is as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidyl, c-myc, hemagglutinin or Flag ^TM(CT) sequence generally is expressed as the fusions with coded polypeptide to label for Kodak, New Haven.This label can insert any position in the polypeptide, comprises carboxyl and N-terminal.

Control region

Term " control region " refer to influence transcribe or translation initiation and speed and transcribe or translation product stability and/or mobile nucleotide sequence.Control region includes but not limited to: promoter sequence, enhancer sequence, response element, albumen recognition site, can induce element, protein binding sequence, 5 ' and 3 ' non-translational region (UTR), transcription initiation site, terminator sequence, polyadenylation sequence and intron.

Term used herein " operability connection " refers to control region and treats that the location of transcription sequence in nucleic acid can allow or help transcribing of this transcription sequence.For example, for encoding sequence being placed promotor control down, the translation initiation site of translation frame that generally makes polypeptide is between the about 1-50 Nucleotide of promotor downstream.Yet promotor can be positioned the translation initiation site upstream up to about 5,000 Nucleotide places, perhaps can be positioned about 2,000 Nucleotide places, transcription initiation site upstream.Promotor generally comprises at least one core (basis) promotor.Promotor also can comprise at least one controlling elements, as enhancer sequence, upstream element or active region, upstream (UAR).For example, suitable enhanser is the cis-regulating element (212 to-154) from octopine synthase (ocs) upstream region of gene district.Fromm etc., The Plant Cell 1:977-984 (1989).Comprise promotor selection depend on several factors, include but not limited to: efficient, selectivity, inducibility, required expression level and cell or tissue-predominant expression.Those skilled in the art usually can be by suitably selecting to regulate with positioning starting and other control region the expression of encoding sequence according to encoding sequence.

Some suitable promotors only or mainly start in some cell type transcribes.For example, can adopt mainly promoters active in germinal tissue (as fruit, ovule, pollen, gynoecium, megagametophyte, ovum, centrocyte, megarchidium, suspensor, synergid, flower, embryo tissue, blastular, embryo, zygote, endosperm, integument or kind skin).Therefore, cell type used herein or tissue-advantage promotor is a variety of priority driven expression promoter in target tissue, but also can produce some expression in other cell type or tissue.The method of identifying and characterize the promoter region in the plant genome DNA for example comprises below with reference to the described method of document: Jordano etc., Plant Cell, 1:855-866 (1989); Bustos etc., Plant Cell, 1:839-854 (1989); Green etc., EMBO J.7,4035-4044 (1988); Meier etc., Plant Cell, 3,309-316 (1991); With Zhang etc., Plant Physiology 110:1069-1079 (1996).

The example of various types of promotors is discussed below.U.S. Patent Application Serial Number 60/505,689; 60/518,075; 60/544,771; 60/558,869; 60/583,691; 60/619,181; 60/637,140; 10/950,321; 10/957,569; 11/058,689; 11/172,703; 11/208,308; With promotors more described below have been described among the PCT/US05/23639 in more detail.Should be understood that promotor can satisfy a kind of criteria for classification according to its activity in a plant species, also can satisfy another kind of criteria for classification according to its activity in another kind of plant species.SEQ ID NO:19-25 has listed the nucleotide sequence of promotor.

The promotor of wide expression

When promotor many, but when not necessarily all promoting to transcribe in the plant tissue, be called " wide expression " promotor.For example, the wide expression promotor can start the transcribing of one or more operability catenation sequences of bud, bud point (top) and leaf, but a little less than the degree that in such as tissues such as root or stems, promotes to transcribe or promote to transcribe.Another example is, the wide expression promotor can promote transcribing of operability catenation sequence in one or more of stem, bud, bud point (top) and leaf, but such as flower or a little less than growing the degree that promotes to transcribe in the germinal tissue such as seed or promote to transcribe.This paper provides the non-limitative example of the wide expression promotor that can comprise in the nucleic acid construct thing to comprise p326 (SEQ ID NO:19), YP0144 (SEQ ID NQ:20), YP0190 (SEQ ID NO:21), p13879 (SEQ ID NO:22), YP0050 (SEQ ID NO:23), p32449 (SEQ ID NO:24), 21876 (SEQ ID NO:25), YP0158, YP0214, YP0380, PT0848 and PTO633 promotor.Other example comprises cauliflower mosaic virus (CaMV) 35S promoter, mannopine synthase (MAS) promotor, available from 1 of the T-DNA of Agrobacterium tumefaciens (Agrobacterium tumefaciens) ' or 2 ' promotor, figwort mosaic virus 34S promotor, actin promoter such as rice actin promoter and ubiquitin promoter such as corn ubiquitin-1 promotor.In some cases, the promotor of wide expression does not comprise the CaMV35S promotor.

The root promotor

The active promotor of root is transcribed as producing in root endodermis, epiblem or the root vascular tissue in root tissue.In some embodiments, the active promotor of root is root-preferred promoter, promptly only or mainly produces in root tissue and transcribes.Root-preferred promoter comprises YP0128, YP0275, PT0625, PT0660, PT0683 and PT0758 promotor.Other root-preferred promoter comprises PT0613, PT0672, PT0678, PT0688 and PT0837 promotor, and these promotors mainly drive in root tissue and transcribe, a little less than the driving effect in ovule and/or seed.Other example of root-advantage promotor comprises the root-specific subdomain (Lam etc. of CaMV35S promotor, Proc.Natl.Acad.Sci.USA86:7890-7894 (1989)), Conlding etc., the root cells specificity promoter and the tobacco RD2 gene promoter of Plant Physiol.93:1203-1211 (1990) report.

Just in the promotor of sophisticated endosperm

In some embodiments, can adopt and just in sophisticated endosperm, driving the promotor transcribe.Just the promoter transcription at sophisticated endosperm generally begins after insemination, take place in the main endosperm tissue during seed development, generally cell in the stage activity the highest.Only is in the active promotor that just has superiority in sophisticated endosperm, but also can adopt the active promotor that also has superiority in other tissue sometimes.This paper provides just comprising at the non-limitative example of the promotor of sophisticated endosperm of can comprising in the nucleic acid construct thing: the rapeseed protein promotor, the Arcelin-5 promotor, Kidney bean protein gene promoter (Bustos etc., Plant Cell 1 (9): 839-853 (1989)), Trypsin inhibitor SBTI promotor (Riggs etc., Plant Cell 1 (6): 609-621 (1989)), ACP promotor (Baerson etc., Plant Mol Biol.22 (2): 255-267 (1993)), stearyl-ACP desaturase gene (Slocombe etc., Plant Physiol 104 (4): 167-176 (1994)), the soybean α subunit promotor (Chen etc. of beta-conglycinin, Proc Natl Acad Sci USA 83:8560-8564 (1986)), oleosin promotor (Hong etc., Plant Mol Biol 34 (3): 549-555 (1997)) and the zein promotor, as 15kD zein promotor, 16kD zein promotor, 19kD zein promotor, 22kD zein promotor and 27kD zein promotor.Other suitable promotor is Osgt-1 promotor (Zheng etc., Mol Cell Biol.13:5829-5842 (1993)), beta-amylase gene promoter and the hordein gene promotor of rice gluten-1 gene.Other just comprises YP0092, PT0676 and PT0708 promotor in the promotor of sophisticated endosperm.

Ovary is organized promotor

Also can adopt promoters active in ovary tissue such as ovule wall and mesocarp, as polygalacturonase promotor, banana TRX promotor and muskmelon actin promoter.

Blastular/early stage endosperm promotor

Express in order to be implemented in blastular/early embryo Ruzhong, can adopt in polar core and/or centrocyte or in polar core precursor (precursor) has activity, but in ovum or ovum precursor the regulatory region of non-activity.Most of suitable promotors are only or mainly to drive expression promoter in polar core or its precursor and/or centrocyte.Also can the transcriptional profile that extends to early stage endosperm development from polar core, find blastular/early stage endosperm-advantage promotor, but the cell stage or transcribe general remarkable reduction late period afterwards in the endosperm development therebetween.Expression in the zygote or the embryo of growing is general to have nothing to do with blastular/early stage endosperm promotor.

The suitable promotor of possibility comprises the promotor derived from following gene: Arabidopis thaliana viviparous-1 (referring to GenBank numbering U93215); Arabidopis thaliana atmycl is (referring to Urao (1996) Plant Mol.Biol, 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopis thaliana FIE (GenBank numbers AF129516); Arabidopis thaliana MEA; Arabidopis thaliana FIS2 (GenBank numbers AF096096); With FIE 1.1 (United States Patent (USP) 6,906,244).Other suitable promotor of possibility comprises the promotor derived from following gene: corn MAC1 is (referring to Sheridan (1996) Genetics, 142:1009-1020); Corn C at3 is (referring to GenBank numbering L05934; Abler (1993) PlantMol.Biol, 22:10131-1038).Other promotor comprises following arabidopsis thaliana promoter: YP0039, YP0101, YP0102, YP0110, YP0117, YP0119, YP0137, DME, YP0285 and YP0212.Other promotor that can adopt comprises following rice promotor: p530c10, pOsFIE2-2, pOsMEA, pOsYp102 and pOsYp285.

The embryo promotor

The control region of transcribing that advantage drives in the after fertilization zygote cell can provide the embryo predominant expression.Only promotor is that advantage drives the promotor of transcribing in the early embryo before heart stage, but drives late period and just the expression promoter in sophisticated embryo is also suitable.Embryo advantage promotor comprises barley lipid transfer protein (Ltp1) promotor (Plant Cell Rep (2001) 20:647-654).

The promotor of organizing that photosynthetic activity is arranged

Have photosynthetic activity organize promotor in photosynthetic activity tissue such as leaf and stem, to produce to transcribe.Most of suitable promotors are only or mainly to drive expression promoter in these tissues.The example of this promotor comprises ribulose-1,5-bisphosphate, the RbcS promotor of 5-bisphosphate carboxylase (RbcS) promotor such as American Larch (Larix laricina), pine tree cab6 promotor (Yamamoto etc., Plant Cell Physiol.35:773-778 (1994)), Cab-1 gene promoter (the Fejes etc. of wheat, Plant Mol.Biol 15:921-932 (1990)), CAB-1 promotor (the Lubberstedt etc. of spinach, Plant Physiol.104:997-1006 (1994)), cab1R promotor (the Luan etc. of paddy rice, Plant Cell4:971-981 (1992)), pyruvic acid orthophosphoric acid salt two kinases (PPDK) promotor (Matsuoka etc. of corn, Proc.Natl.Acad.Sci.U.S.A.90:9586-9590 (1993)), tobacco Lhcb1*2 promotor (Cerdan etc., PlantMol.Biol.33:245-255 (1997)), quasi-sac film protein promotor (the psaD of Arabidopis thaliana (Arahidopsis thaliana) SUC2 sucrose-H+ co-transport promotor (Truernit etc., Planta.196:564-570 (1995)) and spinach, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS).

Inducible promoter

Inducible promoter produces in response to external stimulus such as chemical substance or environmental stimulus and transcribes.For example, inducible promoter can produce in response to hormone such as gibberic acid or ethene or in response to illumination or arid and transcribe.

The basis promotor

The basis promotor is that the necessary minmal sequence of required transcription complex is transcribed in the assembling startup.The basis promotor often comprises " TATA frame " element, can be positioned between the about 15-35 Nucleotide of transcription initiation site upstream.The basis promotor also can comprise " CCAAT box " element (generally being sequence C CAAT) and/or GGGCG sequence, and it can be positioned between the about 40-200 Nucleotide of transcription initiation site upstream, between generally about 60-120 Nucleotide.

Other promotor

The promotor of other type includes but not limited to: the promotor of leaf-advantage, stem/bud-advantage, callus-advantage and aging-advantage.Also can adopt the promotor that is called YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YP0119 and YP0096, described in above-mentioned patent application.

Other control region

Can comprise 5 ' non-translational region (UTR) in the nucleic acid construct thing as herein described.5 ' UTR is transcribed but is not translated, and this non-translational region can comprise+1 Nucleotide between transcription initiation site and translation initiation codon.3 ' UTR can be between translation stop codon and transcript end.UTR can have special function, as improving mRNA courier's stability or reduction translation.The example of 3 ' UTR includes but not limited to polyadenylation signal and transcription termination sequence, as nopaline synthase terminator sequence.

Should be understood that in the recombination of polynucleotide to have more than one control regions, as intron, enhanser, active region, upstream, transcription terminator with induce element.Therefore, more than one control region operability are connected in the polynucleotide sequence of coding carbon regulatory polypeptide.

Transgenic plant and vegetable cell

The present invention also provides transgenic plant cells and the plant that contains at least a recombinant nucleic acid construction described herein.Can transform by construction being integrated into genome, can stable conversion plant or vegetable cell.The cell of stable conversion generally can both keep the nucleic acid of introducing in each cell fission.But also this plant of instantaneous conversion or vegetable cell are so that make the construction unconformability go into its genome.The cell of instantaneous conversion generally can be lost all or part of of the nucleic acid of introducing in each cell fission, so that can not detect the nucleic acid of introducing in daughter cell after the cell fission of enough number of times.Instantaneous conversion all can be used for methods described herein with transgenic plant stable conversion and vegetable cell.

The used transgenic plant cells of methods described herein has constituted part or all of whole plants.The available mode that is suitable for the species of studying is cultivated described plant in incubator, greenhouse or field.Can be according to specific purpose, as recombinant nucleic acid being introduced other strain, recombinant nucleic acid is transferred to other species or is further selected the needs of other required proterties to cultivate transgenic plant.Perhaps, for the transgenic plant kind that adapts to this technology, can carry out vegetative propagation.Filial generation comprises the filial generation of specified plant or plant lines.The filial generation of existing plant comprises F ₁, F ₂, F ₃, F ₄, F ₅, F ₆And progeny plant, or BC ₁, BC ₂, BC ₃With the seed of progeny plant formation, or F ₁BC ₁, F ₁BC ₂, F ₁BC ₃Seed with progeny plant formation.Code name F ₁The hybrid generation that refers to two kinds of different on genetics parental generations.Code name F ₂, F ₃, F ₄, F ₅And F ₆Refer to F ₁The self-pollination of plant or the filial generation of adelphogamy.Can cultivate the seed that transgenic plant produce, make its self-pollination (or outcross and self-pollination) then, to obtain the seed that the nucleic acid construct thing isozygotys.

Can in suspension culture or tissue or organ cultures, cultivate transgenic plant.For the object of the invention, can adopt solid and/or liquid tissue culture technique.When adopting solid medium, transgenic plant cells directly can be placed on the substratum, maybe can be placed on the filter membrane of contact substratum.When adopting liquid nutrient medium, transgenic plant cells can be placed on floatation device, on the porous-film as the contact liq substratum.Generally prepare solid medium by liquid nutrient medium by adding agar.For example, solid medium can be contain the growth hormone of agar and suitable concn such as 2,4 dichloro benzene ethoxyacetic acid (2,4-D) and the phytokinin of suitable concn such as the Murashige and Skoog (MS) substratum of kinetin.

When adopting the vegetable cell of transient transfection, can comprise in the method for transformation that coding has the report sequence of the active report polypeptide of reporter, can detect the active or expression of reporter the opportune moment after conversion.Generally be to transform back about 1-21 days the opportune moment of detecting, 1-14 days according to appointment, about 1-7 days or about 1-3 days.For carry out real-time analysis in different plant species, the expression of the allos nitrogen regulatory polypeptide that checking is not expressed in concrete recipient cell before maybe will verifying adopts instantaneous measurement convenient especially.

Known in the art nucleic acid is introduced the technology of monocotyledons and dicotyledons, include but not limited to: conversion, electroporation and the particle gun of agrobacterium-mediated conversion, virus vector mediation transform, as United States Patent (USP) 5,538,880; 5,204,253; 6,329,571 and 6,013,863 is described.If cell or cultured tissue are as the receptor tissue that transforms, available those skilled in the art's known technology is by the culture aftergrowth (if desired) that transforms.

Floristics

Polynucleotide as herein described and carrier can be used for transforming many unifacial leaves and dicotyledons and vegetable cell system, comprise dicotyledons, as clover, three-coloured amaranth, apple, beans (comprises Kidney bean, lima bean, dried beans, green soya bean), Cauliflower, wild cabbage, Radix Dauci Sativae, Semen Ricini, cherry, garbanzo, witloof, trefoil, cocoa, coffee, cotton, Crambe, flax, grape, shaddock, lemon; root of Szemao crotalaria; lettuce; linseed oil; mango; melon (as watermelon; hami melon); leaf mustard; orange; peach; peanut; pears; pea; pepper; plum; potato; rape; Semen Brassicae campestris (high erucic acid and rapeseed oil); safflower; sesame; soybean; spinach; strawberry; beet; Sunflower Receptacle; sweet potato; tea; tomato and Chinese yam, and monocotyledons such as banana; barley; annual bluegrass; nipa palm; fescue grass; field corn; garlic; grain; oat; oil palm; onion; pineapple; popcorn; paddy rice; rye; rye grass; Chinese sorghum; arabian cron; sugarcane; sweet corn; switchgrass; thimothy grass and wheat.Also can adopt brown alga, green alga, red algae and microalgae.

Therefore, method and composition as herein described can be used for (for example) and belongs to following purpose dicotyledons: Umbellales (Apiales), Arecales (Arecales), Aristolochiales (Aristochiales), chrysanthemum order (Asterales), meat fringe fruit order (Batales), Campanulales (Campanulales), Capparales (Capparales), Caryophyllales (Caryophyllales), Casuarinales (Casuarinales), Celastrales (Celastrales), Cornales (Cornales), Cucurbitales (Cucurbitales), Diapensiales (Diapensiales), Dilleniales (Dilleniales), teasel root order (Dipsacales), Ebenales (Ebenales), Ericales (Ericales), Eucommiales (Eucomiales), Euphorbiales (Euphorbiales), beans order (Fabales), Fagales (Fagales), Gentianales (Gentianales), Mang ox seedling order (Geraniales), Haloragales (Haloragales), Hamamelidales (Hamamelidales), anistree order (Illiciales), Juglandales (Juglandales), Lip shape order (Lamiales), Laurales (Laurales), Lecythidales (Lecythidales), silver hair wooden order (Leitneriales), Linales (Linales), Magnoliales (Magnoliales), Malvales (Malvales), Myricales (Myricales), Myrtales (Myrtales), Nymphaeales (Nymphaeales), Papaverales (Papeverales), Piperales (Piperales), Plantaginales (Plantaginales), Plumbaginales (lumbaginales), Podostemales (Podostemales), Polemoniales (Polemoniales), polygalales (Polygalales), knotweed order (Polygonales), Primulales (Primulales), Proteales (Proteales), Rafflesiales (Rafflesiales), Ranales (Ranunculales), Rhamnales (Rhamnales), Rosales (Rosales), Rubiales (Rubiales), Salicales (Salicales), Santalales (Santales), Sapindales (Sapindales), Sarraceniaceae (Sarraceniaceae), Scrophulariales (Scrophulariales), eggplant order (Solanales), Trochodendrales (Trochodendrales), Theales (Theales), Umbellales (Umbellales), Urticales (Urticales) and Violales (Violales).Method and composition as herein described also can be used for (for example) and belongs to following purpose monocotyledons: Alismatales (Alismatales), Arales (Arales), palm order (Arecales), Asparagales (Asparagales), Bromeliales (Bromeliales), Commelinales (Commelinales), ring flowers and plants orders (Cyclanthales), Cyperales (Cyperales), Eriocaulales (Eriocaulales), Hydrocharitales (Hydrocharitales), Juncales (Juncales), Liliales (Liliales), Najadales (Najadales), blue order (Orchidales), pandanales (Pandanales), Poales (Poales), Restionales (Restionales), Triuridales (Triuridales), Typhales (Typhales), ginger order (Zingiberales) and belong to Gymnospermae is as Cycadales (Cycadales), Ginkgoales (Ginkgoales), the plant of Gnetales (Gnetales) and loose China fir order (Pinales).

Described method and composition can be used for a big class plant species, comprises the species that following dicotyledons belongs to: Amaranthus (Amaranthus), Arachis, Btassica, calendulin (Calendula), Camellia (Camellia), Capsicum (Capsicum), safflower belongs to (Carthamus), olecranon Macroptilium (Cicer), Cichorium (Chicorium), Cinnamomum (Cinnamomum), both citrus (Citrus), Citrullus (Citrullus), Coffea (Coffea), two joint shepherd's purses belong to (Crambe), Cucumis (Cucumis), Cucurbita (Cucurbita), Daucus (Daucus), Wild yam (Dioscorea), Fragaria (Fragaria), Glycine, Gossypium, Helianthus, Lactuca, Lens culinaris belongs to (Lens), linum (Linum), tomato belongs to, Malus (Malus), Du fruit belongs to (Mangifera), Medicago, Mentha (Mentha), Nicotiana (Nicotiana), Ocimum (Ocimum), Olea (Olea), Phaseolus (Phaseolus), pistache (Pistacia), Pisum, Prunus (Prunus), pear (Pyrus), Rosmarinus (Rosmarinus), Salvia (Salvia), flax belongs to (Sesamum), Solanum, spinach belongs to (Spinacia), Theobroma (Theobroma), Thymus (Thymus), Trifolium (Trifolium), genus vaccinium (Vaccinium), Vigna (Vigna) and Vitis; And the kind of following monocotyledons genus: allium (Allium), Ananas (Ananus), Asparagus (Asparagus), Avena (Avena), turmeric (Curcuma), oil palm belongs to (Elaeis), festuca (Festuca), Hordeum (Hordeum), Lemna (Lemna), lolium (Lolium), Musa (Musa), Oryza (Oryza), Panicum (Panicum), Pennisetum (Pennisetum), ladder forage spp (Phleum), annual bluegrass belongs to (Poa), saccharum (Saccharum), Secale (Secale), sorghum (Sorghum), triticale belongs to (Triticosecale), Triticum (Triticum) and Zea (Zea).

Methods described herein and composition also can be used for brown alga, as bladder wrack (Ascophyllum nodosum), black wrack (Fucus vesiculosus), tip edge black wrack (Fucus serratus), extension extra large bar algae (Himanthalia elongate) and wakame (Undaria pinnatifida); Red algae is as wrinkle leaf carrageen (Chondrus crispus), fragrant plant mentioned in ancient texts (Gracilaria verrucosa), navel shape laver (Porphyra umbilicalis) and dulse (Palmariapalmata); Green alga is as Enteromorpha (Enteromorpha spp.) and sea lettuce (Ulva spp.); And little algae, as spirulina (Spirulina sp.) (spirulina plalensis (S.platensis) and spirulina maxim (S.maxima)) and long-eared odontoid algae (Odontella aurita).In addition, this method and composition also can be used for Kou Shi Crypthecodinium cohnii (Crypthecodiniumcohnii), split the kettle algae (Schizochytrium spp.) and rain green blood ball algae (Haematococcus pluvials).

In some embodiments, plant is the member of one of following species: pineapple (Ananus comosus), rape (Brassica campestris), colea (Brassica napus), wild cabbage (Brassica oleracea), soybean (Glycinemax), cotton (Gossypium spp.), lettuce (Lactuca sativa), tomato (Lycopersicon esculentum), powder bajiao banana (Musa paradisiaca), rice (Oryza sativa), potato (Solanum tuberosum), common wheat (Triticum aestivum), grape (Vitis vinifera) or Zea mays (Zea mays).

Suppress the method that the nitrogen regulatory polypeptide is expressed

Polynucleotide described herein and recombinant vectors are used in expresses the nitrogen regulatory polypeptide or suppresses its expression in the interested plant species.Term ＂ expresses ＂ and refers to polynucleotide genetic information by being converted into RNA and being converted into proteinic process by translation mRNA on rrna by catalytic the transcribing of RNA polymerase.＂ raises ＂ or ＂ and activates the regulating and controlling effect that ＂ refers to improve with respect to basis or native state the output of expression product (mRNA, polypeptide or the two), and ＂ downward modulation ＂ or ＂ suppress the regulating and controlling effect that ＂ refers to reduce with respect to basis or native state the output of expression product (mRNA, polypeptide or the two).

Can adopt many methods, comprise that the RNA cutting of sense-rna, ribozyme orientation and RNA disturb (RNAi) to suppress protein expression in the plant based on nucleic acid.Antisense technology is a kind of method of knowing.In this method, the clone is connected in promotor from the nucleic acid sections and the operability of endogenous gene, so that the antisense strand of transcribe rna.Then, as mentioned above recombinant vectors is transformed in the plant, produces the antisense strand of RNA.The nucleic acid sections needs not be the whole sequence of waiting to suppress endogenous gene, but that general and at least a portion waits to suppress endogenous gene is basic identical.Usually, the sequence of use is short more, can use higher homology compensation.The general sequence (as at least 40,50,80,100,200,500 Nucleotide or more) that adopts at least 30 Nucleotide.

Therefore, for example, isolating nucleic acid provided herein can be a coding nitrogen regulatory polypeptide, as the antisense nucleic acid of a kind of above-mentioned nucleic acid of SEQ ID NO:2, SEQ ID NO:4-18 or consensus sequence shown in Figure 1.Can reduce nitrogen regulatory polypeptide encoding gene transcribe or the transcribed nucleic acid of translation product level is the antisense nucleic acid similar or identical with the just encoding sequence of nitrogen regulatory polypeptide.Perhaps, the transcription product of isolating nucleic acid may be similar or identical with the just encoding sequence of nitrogen regulatory polypeptide, but it is the RNA of polyadenylation not, lacks 5 ' cap structure, or contain can not montage intron.

In other method, nucleic acid can be transcribed into can influence ribozyme or the catalytic RNA that mRNA expresses.(referring to U.S. Patent number 6,423,885).Can design ribozyme, make it can and on the specificity site, cut the phosphodiester bond skeleton with any target RNA-specific pairing basically, thereby make the functional inactivation of this target RNA.The heterologous nucleic acids codified is designed for the ribozyme of the specific mRNA transcript of cutting, thereby prevents polypeptide expression.Hammerhead ribozyme can be used for destroying specific mRNA, although can adopt the various ribozymes of cutting mRNA on the specific recognition sequence of site.Hammerhead ribozyme cuts mRNA on the position forming shown in the right side joint district of complementary base with said target mrna.Unique requirement be target RNA contain 5 '-UG-3 ' nucleotide sequence.The structure of hammerhead ribozyme and generation are known in the art.Referring to for example, U.S. Patent number 5,254,678 and WO 02/46449 and the reference wherein quoted.The hammerhead ribozyme sequence can embed in stable RNA such as the transfer RNA (tRNA), to improve cutting efficiency in the body.Perriman etc., Proc.Natl.Acad.Sci.USA, 92 (13): 6175-6179 (1995); De Feyter and Gaudron, " molecular biology method " (Methods in Molecular Biology), the 74th volume, the 43rd chapter, " ribozyme expression in plant " (ExpressingRibozymes in Plants), Turner, P.C compiles, Humana Press Lie., Totowa, NJ.Can adopt naturally occurring RNA endoribonuclease among RNA endoribonuclease such as the tetrahymena thermophila (Tetrahymena thermophila), Cech and colleague carry out detailed description to it.Referring to for example, U.S. Patent number 4,987,071.

Can adopt the method for disturbing (RNAi) based on RNA.It is the cytological mechanism of regulatory gene expression and virus replication that RNA disturbs.It is believed that this mechanism is numerator mediated by double-chain small disturbance RNA.Cell is to destroy the endogenous RNA that the double-stranded RNA sequence is identical therewith to the reaction of this double-stranded RNA.Those skilled in the art will know that the method that designs and prepare RNA interfering; Referring to for example, WO 99/32619 and WO 01/75164.For example, can prepare the construction that comprises the sequence that can be transcribed into RNA interfering.This RNA can be can with annealed RNA own, as have the double-stranded RNA of loop-stem structure.A chain of the stem portion of double-stranded RNA comprises the sequence similar or identical with the just encoding sequence of polypeptide of interest, and the length of this sequence is about 10-2,500 Nucleotide.The length of the sequence similar or identical with just encoding sequence can be 10-500 Nucleotide, a 15-300 Nucleotide, a 20-100 Nucleotide or 25-100 Nucleotide.Another chain of the stem portion of double-stranded RNA comprises the antisense sequences of nitrogen regulatory polypeptide interested, compares with the length of corresponding just sequence, and its length can lack, identical or longer.The length of the loop section of double-stranded RNA can be 10-5,000 Nucleotide, and as 15-1,000 Nucleotide, a 20-500 Nucleotide, perhaps 25-200 Nucleotide.The loop section of RNA can comprise intron.Referring to for example, WO99/53050.

Be used for suppressing the method based on nucleic acid of gene expression in plants at some, suitable nucleic acid can be nucleic acid analog.Nucleic acid analog is modified on base portion, sugar moieties or phosphoric acid skeleton, to improve stability, hybridization ability or the solubleness of (for example) nucleic acid.The modification of base portion comprise deoxyuridine replace deoxythymidine and 5-methyl-2 '-Deoxyribose cytidine and 5-bromo-2 '-Deoxyribose cytidine replaces Deoxyribose cytidine.The modification of sugar moieties comprise 2 ' hydroxyl of modifying ribose with form 2 '-O-methyl or 2 '-O-allyl group sugar.The deoxyribose phosphate skeleton be can modify and morpholino nucleic acid (wherein each base portion is connected in hexa-atomic morpholino ring) or peptide nucleic acid(PNA) (wherein deoxidation phosphoric acid skeleton is replaced by false peptide main chain, and keeps 4 kinds of bases) produced.Referring to for example, Summerton and Weller, 1997, Antisense Nucleic AcidDrugDev., 7:187-195; Hyrup etc., 1996, Bioorgan.Med.Chem., 4:5-23.In addition, deoxidation phosphoric acid skeleton can be replaced by (for example) thiophosphoric acid or phosphorodithioic acid skeleton, the inferior acid amides of phosphoric acid or alkyl phosphotriester main chain.

The transgenic plant phenotype

Can be by specific trait or the activity of selecting or screening engineered plant material, as the proterties or the activity of marker gene or antibiotics resistance gene coding, identify and separate transformant, callus, tissue or plant.Those of ordinary skills know this screening and system of selection.In addition, available physical and biochemical method are identified transformant.These methods comprise Southern analysis or the pcr amplification that is used to detect polynucleotide; Be used to detect Northern trace, S1RNA enzyme protection, primer extension or the RT-PCR amplification of rna transcription thing; Be used to detect the enzymic activity of polypeptide or polynucleotide or the zymetology test of ribozyme activity; And the gel electrophoresis of protein, Western trace, immunoprecipitation and the enzyme linked immune assay that are used to detect polypeptide.Also can adopt other technology such as in situ hybridization, enzyme dyeing and immunostaining to detect the existence or the expression of polypeptide and/or polynucleotide.Carrying out the method for all these technology all knows.

Compare with the corresponding control plant that lacks transgenosis or non-express transgenic, transgenic plant can have the phenotype of change.When polypeptide is expressed, can influence the phenotype of plant in for example suitable time, suitable tissue or with suitable expression level in plant (as transgenic plant).Can be with respect to the control plant of not expressing interested exogenous polynucleotide, as the corresponding wild-type plant, do not change interested exogenous polynucleotide over to but the corresponding plant identical or reduce, suppress or do not induce the identical corresponding plant of genetic background of expression of polypeptides (being in as expression under the control of inducible promoter) with interested transgenic plant genetic background, estimate the phenotype influence.The polypeptide amount that produces when plant or the mRNA amount of this polypeptide of encoding are below 10% of plant interested institute generation, when following, claim this plant " not express " this polypeptide as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01% or 0.001%.Available following method evaluation expression for example comprises: RT-PCR, Northern trace, S1RNA enzyme protection, primer extension, Western trace, gel electrophoresis of protein, immunoprecipitation, enzyme linked immune assay, chip test and mass spectrum.It should be noted that if polypeptide is expressed under the control of the promotor of tissue specificity or wide expression, can be in whole plants or selected tissue evaluation expression.Similarly,, express, then can on required time, express by selective evaluation as the specified time in growth or after inducing if polypeptide is expressed at specified time.

In some embodiments, the nitrogen regulatory polypeptide is expressed the seed nitrogen level that the plant that is conditioned can have raising.For example, nitrogen regulatory polypeptide described herein can be expressed in transgenic plant, causes the seed nitrogen level to raise.Compare with the seed nitrogen level of not expressing this genetically modified corresponding control plant, the seed nitrogen level can raise at least 5%, as 5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100% or greater than 100%.In some embodiments, the nitrogen regulatory polypeptide is expressed the seed nitrogen level that the plant that is conditioned can have reduction.Compare with the seed nitrogen level of not expressing this genetically modified corresponding control plant, the seed nitrogen level can reduce at least 5%, as 5,10,15,20,25,30,35,40,45,50% or greater than 50%.

Regulating the seed nitrogen level may include but not limited to its useful plant: clover, lettuce, Radix Dauci Sativae, onion, Cauliflower, tomato, potato, sugarcane, grape, cotton, rapeseed oil (canola), sweet corn, popcorn, field corn, pea, beans, safflower, soybean, coffee, three-coloured amaranth, Semen Brassicae campestris, peanut, Sunflower Receptacle, oil palm, wheat, rye, barley, oat, paddy rice, grain, strawberry, pineapple, melon, peach, pears, apple, cherry, orange, lemon, shaddock, plum, mango, banana, fescue grass, rye grass, annual bluegrass, trifolium, thimothy grass, arabian cron, switchgrass and Chinese sorghum.The increase of seed nitrogen can improve the usually insufficient geographic nutrition supply of protein/amino acid whose dietary intake in these plants.Seed nitrogen reduces that to can be used for seed be not the situation that results are used for the main plant part of the mankind or animals consuming in these plants.

In some embodiments, the nitrogen regulatory polypeptide is expressed one or more non-seed tissues of the plant of being regulated, can improve or reduce as nitrogen level in leaf texture, stem tissue, root or bulb tissue or the fruit tissue beyond the seed.For example, compare with the seed nitrogen level of not expressing this genetically modified corresponding control plant, nitrogen level can improve at least 5%, as 5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100% or greater than 100%.In some embodiments, the nitrogen regulatory polypeptide nitrogen level of expressing in one or more non-seed tissues of the plant regulated can reduce.Compare with the seed nitrogen level of not expressing this genetically modified corresponding control plant, nitrogen level can reduce by 5%, as 5,10,15,20,25,30,35,40,45,50 or greater than 50%.

The nitrogen level of regulating non-seed tissue may include but not limited to its useful plant: clover, lettuce, Radix Dauci Sativae, onion, Cauliflower, tomato, potato, sugarcane, grape, sweet corn, popcorn, field corn, pea, beans, safflower, soybean, coffee, three-coloured amaranth, Semen Brassicae campestris, peanut, Sunflower Receptacle, oil palm, wheat, rye, barley, oat, paddy rice, grain, strawberry, pineapple, melon, peach, pears, apple, cherry, orange, lemon, shaddock, plum, mango, banana, fescue grass, rye grass, annual bluegrass, trifolium, thimothy grass, arabian cron, switchgrass and Chinese sorghum.

The increase of non-seed nitrogen can improve the nutrient contg in edible fruits and the vegetables in these plants, or improves animal-feed.Non-seed nitrogen reduces can make nitrogen more effectively be assigned to the plant part that results are used for the mankind or animals consuming.

Usually, with suitable parameters or nonparametric statistics, as X ²Check, Student ' s t-check, Mann-Whitney check or F-check record p≤0.05 o'clock, think that transgenic plant or cell have significance,statistical with respect to the nitrogen content difference (as increasing) of control plant or cell.In some embodiments, p＜0.01, p＜0.005 or p＜0.001 o'clock, the difference of nitrogen content has significance,statistical.The cell of nitrogen content and control plant has statistical significant difference to show that the recombinant nucleic acid that exists in (1) transgenic plant causes nitrogen level to change and/or (2) should further study recombinant nucleic acid as the material standed for that changes the plant nitrogen content in the seed of transgenic plant.

Goods

This paper also provides and can comprise (for example) goods from the seed mixture (as the seed mixture of basic homogeneous) of transgenic plant that this paper provides.The seed mixture is adjusted and packed to available mode known in the art, with the preparation goods.The packing of seed can have label,, as be fixed on mark or label on the wrapping material, be printed on the label on the wrapping material or insert label in the packing.Can illustrate in the label that the plant that contained seed grows in the packing can produce the crop that the seed nitrogen level is higher than corresponding control plant thus.

Further describe the present invention in following examples, these embodiment can not limit the described scope of the invention of claims.

Embodiment

Embodiment 1-transgenic plant

Adopt following symbol: T among the embodiment ₁: first-generation transformant; T ₂: the s-generation, autophilous T ₁The filial generation of plant; T ₃: the third generation, autophilous T ₂The filial generation of plant; T ₄: the 4th generation, autophilous T ₃The filial generation of plant.To independently transform and be called incident.

Following separate nucleic acid is from Arabidopis thaliana (Arabidopsis thaliana) environmental Wassilewskija (Ws) plant.Ceres cDNA ID 2998984 (SEQ ID NO:1) is that expectation coding 505 amino acid whose (SEQ ID NO:2) is inferred the genomic dna cloning that oligopeptides is transported polypeptide.Ceres clones 117581 (cDNA ID 23364185; SEQID NO:3) be that expectation coding 587 amino acid whose (SEQ ID NO:4) is inferred the cDNA clone of peptide transhipment polypeptide.

The above-mentioned polynucleotide that respectively separate are cloned in the carrier that contains glufosinates acetyltransferase gene, and this carrier can make plant transformed produce Finale ^TMResistance.Structure contains operability and is connected in the Ceres cDNA ID 2998984 of cauliflower mosaic virus (CaMV) 35S control region or Ceres clone 117581 the two carriers of NB42-35S.The derivative that the two carriers of NB42-35S are the two carriers of pMOG800.

Transform the environmental C24 plant of wild-type Arabidopis thaliana separately with the two carriers of the NB42-35S that contains Ceres cDNA ID 2998984 or Ceres clone 117581.Basically according to Bechtold etc., CR.Acad.Sci.Paris, the described conversion of 316:1194-1199 (1993).

The transgenic arabidopsis strain that contains Ceres cDNA ID 2998984 or Ceres clone 117581 is called SR00829 and SR05001.Pass through Finale ^TMResistance, carry out polymerase chain reaction (PCR) amplification and the PCR product has been carried out sequence verification having Ceres cDNA ID 2998984 carriers among the SR00829, and SR05001 exists Ceres to clone 117581 carriers by the greenery tissue extract.As the contrast of the environmental C24 plant of transgenic arabidopsis, transform the environmental C24 plant of wild-type Arabidopis thaliana with empty carrier NB42-35S.

Nucleotide sequence in Ceres cDNA ID 2998984 and Ceres clone's 117581 the plant and the environmental Columbia sequence of homology Arabidopis thaliana are made comparisons.Nucleotide sequence and homology Columbia sequence differs from a Nucleotide in Ceres clone 117581 the plant.Compare with homology Columbia sequence, nucleotide sequence contains base pair insertion, disappearance and replacement in the plant of Ceres cDNA ID2998984.

For whether the overall structure of determining the prediction polypeptide that Ceres cDNA ID 2998984 encodes is similar to the nucleotide sequence coded prediction polypeptide of homology Columbia, analyze transhipment peptide characteristic membrane spaning domain possible in these two sequences with Tmpred program (ch.embnet.org/software/TMPRED_form).Analysis revealed, these two kinds prediction polypeptide have 12 of expection and stride the film district.Yet, the prediction of translation initiation site is shown the N-terminal of the prediction polypeptide of Ceres cDNA ID 2998984 codings is than short 40 amino acid of the prediction polypeptide of Columbia homologue coding.Therefore, according to SignalP algorithm (cbs.dtu.dk/services/SignalP/), the prediction polypeptide of Ceres cDNA ID 2998984 codings lacks the contained secretion signal peptide sequence of preceding 40 amino acid of the Columbia peptide sequence of prediction.The predicted polypeptide sequence of analyzing Ceres cDNA ID 2998984 codings with iPSORT signal sequence prediction algorithm (hc.ims.u-tokyo.ac.jp/iPSORT/index.html#aaindex) shows that preceding 40 amino acid contain possible chloroplast targeted signal sequence.

Screening transgenic arabidopsis strain as described below: 1) T in the screening greenhouse ₁The form phenotype of material standed for, 2) analysis T ₂The carbon of seed and nitrogen content, 3) checking T ₃Carbon and/or nitrogen content raising and 4 in the seed) estimates T ₂Negative phenotype and the Finale of plant ^TMSeparate.

Each five incident of screening SR00829 and SR05001 are at T ₁The visible phenotypic that produces in generation changes.All T ₁The barment tag of plant is identical with corresponding control plant.

The analysis of carbon and nitrogen content in embodiment 2-transgenic arabidopsis seed

About 2.00 ± 0.15mg the exsiccant of weighing transgenic arabidopsis seed (about 100) adds in the tin can carbon of analyzing total and nitrogen content.Prepare three kinds of coupling contrasts in the mode identical with test sample, even at interval in whole batch.In each batch first three sample is respectively blank sample (empty tin can), bypass (bypass) (about 5mg aspartic acid) and standard model (5.00 ± 0.15mg aspartic acid).Add in the tin can with analytical balance weighing aspartic acid.Insert a blank sample between per 15 test samples.

(Thermo Finnigan, San Jose CA) finish analysis with the FlashEA1112NC analyser.Device parameter is as follows: 900 ℃ in left stove, 840 ℃ in right stove, 50 ℃ in baking oven, airflow carrier 130mL/ minute, air-flow reference 100mL/ minute.The data parameters LLOD of standard model is 0.25mg, other material difference.The data parameters LLOQ of standard model is 3mg, and the data parameters LLOQ of seed tissue is 1mg, other material difference.

Carry out quantitatively with EA1112 software.Make result standardization, and represent with absolute percent.Analyze each sample in triplicate, base of calculation is poor.The total carbon content that recorded the non-transgenic contrast in the past is 53.3 ± 2.4%, and total nitrogen content is 3.9 ± 0.3%.The theoretical standard difference of aspartic acid standard substance is carbon ± 2.0%, nitrogen ± 1.0%.In order to be called as effectively, needing aspartic acid (standard substance) weight of each wheel is 5mg ± 0.15mg, needs the nitrogen of blank sample or carbon content to be recorded as zero.Need the standard deviation percentage ratio between the repeat samples to be lower than 10%.

Embodiment 3-SR00829 event result

The SR00829 T after twice incident that contains Ceres cDNA ID 2998984 as analysis as described in the embodiment 2 ₂And T ₃Total carbon in the seed and total nitrogen content.

Compare the T of SR00829 after twice incident with the nitrogen content of corresponding contrast seed ₂The nitrogen content of seed significantly increases.As shown in table 1, compare with the nitrogen content of contrast seed, nitrogen content is increased to 110% and 109% in the seed of incident-01 and-02.

The T of table 1:SR00829 incident ₂And T ₃Seed total nitrogen content (% contrast)

	Incident-01	Incident-02	Contrast
	Incident-01	Incident-02	Contrast	T ₂	110±1	109±2	100±1
The p value	0.001	0.002	NA	T ₂	110±1	109±2	100±1
The p value	0.001	0.002	NA	T ₃	111±3	118±5	100±3
The p value	<0.01	0.01	NA	T ₃	111±3	118±5	100±3

Compare with the nitrogen content of corresponding contrast seed, SR00829 is T after twice incident ₃The nitrogen content of seed significantly increases.As shown in table 1, compare with the nitrogen content of contrast seed, nitrogen content is increased to 111% and 118% respectively in the seed of incident-01 and-02.

Observe the T of SR00829 incident ₂And T ₃The carbon content of the carbon content of seed and corresponding contrast seed is significantly difference not.

Be used to analyze the T of the SR00829 incident of carbon and nitrogen content ₃Seed is from a T through each incident ₂Plant is collected.

SR00829 is through the T after the incident-01 and-02 ₂The isolating result of FinaleTM resistance is that resistance and sensitivity ratio are 3:1 in the plant.

T ₂SR00829 and control plant sprout, come into bloom, lotus throne district, fertilizability, plant height and general morphology/configuration aspects all do not have observable or statistical significant difference.

The result of embodiment 4-SR05001 incident

The SR05001 T after twice incident that contains Ceres clone 117581 as analysis as described in the embodiment 2 ₂And T ₃Total carbon in the seed and total nitrogen content.

Compare with the carbon content of corresponding contrast seed, SR05001 is T after an incident ₂The carbon content of seed significantly reduces.As shown in table 2, compare with the carbon content of contrast seed, carbon content is reduced to 97% in the seed of incident-02.

The T of table 2:SR05001 incident ₂And T ₃Seed total carbon content (% contrast)

	Incident-02	Incident-03	Contrast
	Incident-02	Incident-03	Contrast	T ₂	97±2	102±2	100±1
The p value	0.03	0.14	NA	T ₂	97±2	102±2	100±1
The p value	0.03	0.14	NA	T ₃	106±1	107±2	100±2
The p value	0.01	0.01	NA	T ₃	106±1	107±2	100±2

Compare with the nitrogen content of corresponding contrast seed, SR05001 is T after twice incident ₂The nitrogen content of seed significantly increases.As shown in table 3, compare with the nitrogen content of contrast seed, nitrogen content is increased to 112% and 115% respectively in the seed of incident-02 and-03.

The T of table 3:SR05001 incident ₂And T ₃Seed total nitrogen content (% contrast)

	Incident-02	Incident-03	Contrast
	Incident-02	Incident-03	Contrast	T ₂	112±3	115±1	100±4
The p value	<0.01	<0.01	NA	T ₂	112±3	115±1	100±4
The p value	<0.01	<0.01	NA	T ₃	109±1	106±2	100±2
The p value	<0.01	0.01	NA	T ₃	109±1	106±2	100±2

Compare with the carbon content of corresponding contrast seed, SR05001 is T after twice incident ₃The carbon content of seed significantly increases.As shown in table 2, compare with the carbon content of contrast seed, carbon content is increased to 106% and 107% respectively in the seed of incident-02 and-03.

Compare with the nitrogen content of corresponding contrast seed, SR05001 is T after twice incident ₃The nitrogen content of seed significantly increases.As shown in table 3, compare with the nitrogen content of contrast seed, nitrogen content is increased to 109% and 106% respectively in the seed of incident-02 and-03.

Be used to analyze the T of the SR05001 incident of carbon and nitrogen content ₃Seed is from a T through each incident ₂Plant is collected.

SR05001 is through the T after the incident-02 and-03 ₂Finale in the plant ^TMThe isolating result of resistance is that resistance and sensitivity ratio are 3:1.

T ₂SR05001 and control plant sprout, come into bloom, lotus throne district, fertilizability, seed size and general morphology/configuration aspects all do not have observable or statistical significant difference.

Embodiment 5-function homologue and/or straight determining to homologous sequence

If theme and search sequence encoded protein matter have identity function and/or activity, think that then subject nucleotide sequence is the function homologue of search sequence and/or directly to homologue.With being called mutual BLAST (Rivera etc., Proc.Natl.Acad.Sci. (U.S.A.), 95:6239-6244 (1998)) method is identified the public and patent peptide sequence database from available, comprises function homologue possible in NCBI NR Protein Data Bank and Ceres clone's the peptide translation database and/or directly to homologous sequence.Begin before the mutual BLAST method, with BLAST specific inquiry polypeptide of search in all peptides of source species, with the sequence homogeny of identifying and inquire about polypeptide be 80% or higher and comparison length account for 85% or more polypeptide of the shorter sequence of being compared.The polypeptide of inquiry polypeptide and above-mentioned evaluation is called as bunch.

Main mutual BLAST method is made up of the two-wheeled blast search: forward lookup and reverse search.In the forward lookup step, with all protein sequence BLAST retrieval of species interested from the inquiry peptide sequence " polypeptide A " that comes source category SA.With E-value cutoff 10 ^-5Determine the highest hitting with homogeny cutoff 35%.In the highest hitting, the sequence that the E value is minimum is called as the best hits, and is considered to potential function homologue and/or directly to homologue.Hit with the best or the sequence homogeny of initial query polypeptide be 80% or higher any other the highest hitting also be considered to potential function homologue and/or directly to homologue.Kind interested repeats this method to all.In reverse search, be used for the highest the hitting that from all kinds, identify in all protein sequence BLAST retrieval forward lookups of source category SA.If forward lookup the highest hit and above-mentioned bunch polypeptide is returned as its best is hit, also be considered to potential function homologue and/or directly to homologue.

Manual check potential function homologue and/or, to identify the function homologue and/or directly to homologue directly to the homologue sequence.The representative functions of SEQ ID NO:4 is directly seen Fig. 1 to homologue.Function directly sees Table 4 to the homogeny percentage ratio of homologue and SEQ IDNO:4.

Table 4: with Ceres clone's 117581 (SEQ ID NO:4) homogeny percentage ratio

Title	Species	SEQ?ID?NO：	The % homogeny	The e value
Title	Species	SEQ?ID?NO：	The % homogeny	The e value	Ceres clone: 328378	Zea mays	5	70.4	0
gi\|2655098	Barley (Hordeum vulgare) vulgare subspecies	6	68.2	0	Ceres clone: 328378	Zea mays	5	70.4	0
gi\|2655098	Barley (Hordeum vulgare) vulgare subspecies	6	68.2	0	gi\|34895718	Paddy rice Japan subspecies	7	68	0
gi\|50933627	Paddy rice Japan subspecies	8	64	0	gi\|34895718	Paddy rice Japan subspecies	7	68	0
gi\|50933627	Paddy rice Japan subspecies	8	64	0	gi\|4102839	Tomato	9	63	0
gi\|31088360	Broad bean (vicia faba)	10	62.9	0	gi\|4102839	Tomato	9	63	0
gi\|31088360	Broad bean (vicia faba)	10	62.9	0	gi\|6635838	Plum (prunus dulcis)	11	61.4	0
gi\|56784523	Paddy rice Japan subspecies	12	56.7	0	gi\|6635838	Plum (prunus dulcis)	11	61.4	0
gi\|56784523	Paddy rice Japan subspecies	12	56.7	0	gi\|56784524	Paddy rice Japan subspecies	13	51.6	0
gi\|6409176	Paddy rice	15	49.5	0	gi\|56784524	Paddy rice Japan subspecies	13	51.6	0
gi\|6409176	Paddy rice	15	49.5	0	gi\|50059161	Common wheat	14	49.5	0
Ceres clone: 352232	Zea mays	16	48.5	0	gi\|50059161	Common wheat	14	49.5	0
Ceres clone: 352232	Zea mays	16	48.5	0	gi\|33411520	Peach (prunus persica)	17	47.1	0
gi\|31429847	Paddy rice	18	47.1	0	gi\|33411520	Peach (prunus persica)	17	47.1	0

Other embodiment

Though should be understood that with the present invention's detailed description and described the present invention, above-mentioned specification sheets is intended to illustrate and the determined scope of the invention of the scope of unrestricted appended claims.The scope of appended claims comprises others, advantage and modification.

Sequence table

<110>Ceres，Inc.

＜120〉regulate plant nitrogen levels

<130>11696-150WO1

<150>US?60/705,119

<151>2005-08-02

<150>US?60/637,311

<151>2004-12-16

<160>25

<170>FastSEQ?for?Windows?Version?4.0.

<210>1

<211>1446

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(1)..(1446)

＜223〉Ceres cDNAID numbering 2998984

<220>

<221>CDS

<222>(120)..(687)

＜223〉referring to SEQ ID NO:2

<400>1

<210>2

<211>505

<212>PRT

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(50)..(443)

＜223〉Pfam Name:PTR2; Pfam describes: POT family

<400>2

<210>3

<211>1764

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(1)..(1764)

＜223〉Ceres clone ID numbering 117581

<220>

<221>CDS

<222>(1)..(1761)

＜223〉referring to SEQ ID NO:4

<400>3

<210>4

<211>587

<212>PRT

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(96)..(499)

＜223〉Pfam Name:PTR2; Pfam describes: POT family

<400>4

<210>5

<211>580

<212>PRT

＜213〉Zea mays (Zea mays)

<220>

<221>misc_feature

<222>(1)..(580)

＜223〉Ceres clone ID numbering 328378

<400>5

<210>6

<211>579

<212>PRT

＜213〉barley (Hordeum vulgare)

<220>

<221>misc_feature

<222>(1)..(579)

＜223〉public GI numbering 2655098

<400>6

<210>7

<211>580

<212>PRT

＜213〉rice (Oryza sativa) (Japanese cultivar)

<220>

<221>misc_feature

<222>(1)..(580)

＜223〉public GI numbering 34895718

<400>7

<210>8

<211>572

<212>PRT

＜213〉rice (Oryza sativa) (Japanese cultivar)

<220>

<221>misc_feature

<222>(1)..(572)

＜223〉public GI numbering 50933627

<400>8

<210>9

<211>580

<212>PRT

＜213〉tomato (Lycopersicon esculentum)

<220>

<221>misc_feature

<222>(1)..(580)

＜223〉public GI numbering 4102839

<400>9

<210>10

<211>584

<212>PRT

＜213〉broad bean (Vicia faba)

<220>

<221>misc_feature

<222>(1)..(584)

＜223〉public GI numbering 31088360

<400>10

<210>11

<211>559

<212>PRT

＜213〉plum (prunus dulcis)

<220>

<221>misc_feature

<222>(1)..(559)

＜223〉public GI numbering 6635838

<220>

<221>misc_feature

<222>(46)..(46)

＜223〉Xaa is any aa, the unknown or other

<400>11

<210>12

<211>557

<212>PRT

＜213〉rice (Oryza sativa) (Japanese cultivar)

<220>

<221>misc_feature

<222>(1)..(557)

＜223〉public GI numbering 56784523

<400>12

<210>13

<211>545

<212>PRT

＜213〉rice (Oryza sativa) (Japanese cultivar)

<220>

<221>misc_feature

<222>(1)..(545)

＜223〉public GI numbering 56784524

<400>13

<210>14

<211>586

<212>PRT

＜213〉common wheat (Triticum aestivum)

<220>

<221>misc_feature

<222>(1)..(586)

＜223〉public GI numbering 50059161

<400>14

<210>15

<211>584

<212>PRT

＜213〉rice (Oryza sativa)

<220>

<221>misc_feature

<222>(1)..(584)

＜223〉public GI numbering 6409176

<400>15

<210>16

<211>584

<212>PRT

＜213〉Zea mays (Zea mays)

<220>

<221>misc_feature

<222>(1)..(584)

＜223〉Ceres clone ID numbering 352232

<220>

<221>misc_feature

<222>(524)..(524)

＜223〉Xaa is any aa, the unknown or other

<400>16

<210>17

<211>596

<212>PRT

＜213〉peach (prunus persica)

<220>

<221>misc_feature

<222>(1)..(596)

＜223〉public GI numbering 33411520

<400>17

<210>18

<211>576

<212>PRT

＜213〉rice (oryza sativa) (Japanese cultivar)

<220>

<221>misc_feature

<222>(1)..(576)

＜223〉public GI numbering 31429847

<400>18

<210>19

<211>1954

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(1)..(1954)

＜223〉Ceres promotor p326

<400>19

<210>20

<211>881

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(1)..(881)

＜223〉Ceres promotor YP0144

<400>20

<210>21

<211>1002

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(1)..(1002)

＜223〉Ceres promotor YP0190

<400>21

<210>22

<211>1514

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(1)..(1514)

＜223〉Ceres promotor p13879

<400>22

<210>23

<211>1026

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(1)..(1026)

＜223〉Ceres promotor YP0050

<400>23

<210>24

<211>2016

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(1)..(2016)

＜223〉Ceres promotor p32449

<400>24

<210>25

<211>1823

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<220>

<221>misc_feature

<222>(1)..(1823)

＜223〉the Ceres promotor 21876

<400>25

Claims

1. method of regulating nitrogen level in the plant, described method comprises isolating nucleic acid introduced plant cell, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny that is selected from down the aminoacid sequence of organizing: SEQ ID NO:2, SEQ ID NO:4-18, with consensus sequence shown in Figure 1, the nitrogen level in the plant tissue that described vegetable cell produces is variant with the respective horizontal in the control plant tissue that does not contain described nucleic acid.

2. the method for claim 1 is characterized in that, described difference is that nitrogen level raises.

3. the method for claim 1 is characterized in that, described isolating nucleic acid operability is connected in control region.

4. method as claimed in claim 3, it is characterized in that described control region is the promotor that is selected from down group: YP0092, PT0676, PT0708, PT0613, PT0672, PT0678, PT0688, PT0837, the rapeseed protein promotor, the Arcelin-5 promotor, the Kidney bean protein gene promoter, the Trypsin inhibitor SBTI promotor, the ACP promotor, the stearyl-ACP desaturase gene, the soybean α 1 subunit promotor of beta-conglycinin, the oleosin promotor, 15kD zein promotor, 16kD zein promotor, 19kD zein promotor, 22kD zein promotor, 27kD zein promotor, the Osgt-1 promotor, beta-amylase gene promoter and hordein gene promotor.

5. method of producing plant tissue, described method comprises cultivates a kind of vegetable cell that contains isolating nucleic acid, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny that is selected from down the aminoacid sequence of organizing: SEQ ID NO:2, SEQ ID NO:4-18, with consensus sequence shown in Figure 1, the nitrogen level in the described tissue is variant with the respective horizontal in the control plant tissue that does not contain described nucleic acid.

6. vegetable cell that contains isolating nucleic acid, it is 80% or the nucleotide sequence of higher polypeptide that described isolating nucleic acid comprises coding and the sequence homogeny that is selected from down the aminoacid sequence of organizing: SEQ ID NO:2, SEQ ID NO:4-18, with consensus sequence shown in Figure 1, the nitrogen level in the plant tissue that described vegetable cell produces is variant with the respective horizontal in the control plant tissue that does not contain described nucleic acid.

7. transgenic plant, it comprises the described vegetable cell of claim 6.

8. the filial generation of the described plant of claim 7 is characterized in that, the nitrogen level of described filial generation is variant with the nitrogen level in the corresponding control plant that does not contain described isolating nucleic acid.

9. the seed of the described transgenic plant of claim 7.

10. the nutritive issue of the described transgenic plant of claim 7.

11. a food, it comprises the nutritive issue of the described transgenic plant of claim 7.

12. a feeds product, it comprises the nutritive issue of the described transgenic plant of claim 7.