CN107629121A - One from transcription factor ZmNLP9 of corn and application thereof - Google Patents

Abstract

The present invention relates to field of plant genetic, one is provided from transcription factor ZmNLP9 of corn and application thereof, the nucleotide sequence of the transcription factor is as shown in SEQ ID NO.1, the amino acid sequence that it is encoded is as shown in SEQ ID NO.2, arabidopsis after being transferred to using the transcription factor is compared with acceptor arabidopsis, and its fresh weight substantially increases, vivo acid content and total nitrogen content increase, main root length substantially increase.

Description

One from transcription factor ZmNLP9 of corn and application thereof

Technical field

The present invention relates to field of plant genetic, there is provided one from corn transcription factor ZmNLP9 and Its purposes.

Background technology

Nitrogen is one of important mineral nutrient element for maintaining plant growth and biological yield, and is participated in vivo The synthesis of a variety of large biological molecules such as nucleic acid, protein, lipid, chlorophyll.Nitrogen, which lacks, significantly limits growing for plant (Krouk et al.,2010,Current Opinion in Plant Biology 13,265 272).It is so raw in agricultural Crop yield largely is improved using nitrogenous fertilizer extensively in production, improves constantly agriculture production cost.The annual whole world is administered in soil 10,000,000 tons of nitrogenous fertilizer about 8.5-9.0, but wherein 50-70% fails to be absorbed by plants and be lost in environment, causes richness The serious ecological nuclear sterile barleys such as nutrient laden, soil acidity.In order to promote the sustainable development of agricultural, preserve the ecological environment Put into nitrogenous fertilizer is reduced, just have to improve the utilization ratio of plant nitrogen, this has become the heat of current plant nutrient research Point.

Most crops such as wheat, corn are all that nitrate nitrogen is both needed for growth and development of plants to absorb based on nitrate nitrogen The important nutrient wanted, it is that important signaling molecule can regulate and control plant absorbing for nitrate again.But in soil The change in concentration scope of nitrate is very big, and in order to preferably adapt to environment, plant evolution has gone out low affinity nitrate transport protein (NRT1 families) and high affinity nitrate transport protein (NRT2 families).In model plant arabidopsis, NRT1 families have 53 There are 7 members in member, NRT2 families.Wherein NRT1.1 and NRT1.2 are expressed in root, mainly responsible high concentration nitrate condition Under absorption；The members such as NRT1.5-NRT1.12 are then primarily involved in assigning process (Different Organs and the tissue of nitrate nitrogen in vivo Between distribution).In addition, AtNRT1.1 is the nitrate transport protein of parents' sum, and it is proved to be the impression of nitrate nitrogen Device (Transceptor), it adapts to the concentration of nitrate in environment and carries out the conversion of high-affinity and low compatibility, this Transformation relies primarily on the threonine (thereonine, Thr) of 101st of the CBL-CIPK protein kinasis to its protein Phosphorylation state regulate and control come what is realized, this enables plant to adapt to the change of nitrate concentration rapidly and adjust absorption nitric acid The mode of salt.When nitrate concentration is relatively low in environment, many NRT2 members by special induced expression, plant in root so as to improve Absorbability of the thing to nitrate nitrogen.Nitrate can be temporarily stored in root cells vacuole in vivo, can also be with transpiration current by wood Matter portion conduit is transported to overground part；Nitrous acid can be reduced in the presence of nitrate reductase (NR) into internal nitrate anion Root, enter back into chloroplaset or plastid and be reduced to NH⁴⁺, glutamine synthetic reaction is eventually entered into, and pass through transamination Synthesize the amino acid needed for plant.

In model plant arabidopsis, absorption, transhipment and the assimilation process of nitrate and the sound to root system to nitrate Should be by fine and strict regulation and control, these processes are mainly to have the controlling gene during nitrate signal transduction to complete. Verified AtNRT1.1 has regulated and controled turning for nitrate transport gene, nitrate reductase and nitrite reductase as controlling gene Record, so as to regulate and control Nitrogen utility；And AtNRT1.1 is occurred by lateral root under the conditions of influenceing AtANR1 regulation and control nitrate nitrogens；AtNRG2 Positioned at AtNRT1.1 upstream, the utilization of nitrate nitrogen is influenceed by regulating and controlling AtNRT1.1；AtNGR2 can also be with AtNLP7 interaction shadows The adjustment signal of the nitrate nitrogen when ammonium ion be present is rung, the above proves that nitrate nitrogen controlling gene can significantly affect nitrate nitrogen Utilize the response with Nitrate in Plants.

Corn is the third-largest cereal crops in China, and a kind of important feed, economy and bio-energy crop in China, its demand Measure growing day by day.But a large amount of applied nitrogens during China's Maize Production, cause corn very low to the utilization rate of nitrogenous fertilizer (Juetal.,2009,Proc Natl Acad Sci USA,106:3041-3046).The corn variety of high nitrogen effect is cultivated, is Solve the key of the problem and effective way, and the basic demand of agricultural sustainable development and enhancing product competitiveness. But traditional genetic breeding needs a long-term process, can not solve the problem in the short time to a certain extent.And utilize now Genetic engineering means, in particular with the maturation of corn gene technology so that we possibly through genetic engineering means Quickly obtain the efficient kind of nitrogen.And it can fast and effeciently obtain the efficient new varieties of transgenosis nitrogen using genetic engineering means and exist It is heavily dependent on the utilization of good functional gene.

The content of the invention

The present inventor is directed to the situation of above-mentioned prior art, there is provided a transcription factor for deriving from corn ZmNLP9 and application thereof, the nucleotide sequence of the transcription factor is as shown in SEQ ID NO.1, and the amino acid sequence that it is encoded is such as Shown in SEQ ID NO.2, compared with acceptor arabidopsis, its biological fresh weight substantially increases the arabidopsis after being transferred to using the transcription factor Add, vivo acid content and total nitrogen content increase, main root length substantially increases.

This concrete technical scheme of the invention is as follows：

One transcription factor ZmNLP9 for deriving from corn, the nucleotide sequence such as SEQ ID NO.1 institutes of the transcription factor Show, for the amino acid sequence that it is encoded as shown in SEQ ID NO.2, above-mentioned transcription factor ZmNLP9 is that inventor passes through polymerase chain Formula react (PCR) method, using tri-leaf period Leaves of Maize Seedlings extract RNA and by its reverse transcription into cDNA as template, utilization is following Primer amplification obtains：

Sense primer：5 '-GGGTCGACGCAGCAGCAAGGTTTCATCCCAT-3 ', its nucleotide sequence such as SEQ ID Shown in NO.3；

Anti-sense primer：5 '-GGGGTACCACCAGAGCTTCCACAAGAACTGC-3 ', its nucleotide sequence such as SEQ ID Shown in NO.4；

It is transferred to recipient plant by inventor in several ways after above-mentioned transcription factor is obtained, and as a result finds table Plant nitrogen utilization ratio can be significantly improved up to corn ZmNLP9 genes：

With nitrate nitrogen (NO₃ ^-) be only nitrogen source under conditions of, be transferred to the arabidopsis of ZmNLP9 genes and acceptor arabidopsis Compare, its plant fresh weight substantially increases, vivo acid content and total nitrogen content increase, main root length substantially increase.

Based on the discovery, transcription factor provided by the present invention is transferred to by inventor can after recipient plant and overexpression To obtain the plant for including following at least one advantage：

A, biomass is higher than the recipient plant；

B, plant amino acid content, total nitrogen content are higher than the recipient plant；

C, main root length is longer than the recipient plant；

Based on above-mentioned transcription factor ZmNLP9, it can also continue to modify it and import again in recipient plant, with It is expected to reach more preferable expression effect：

1) basis, which is actually needed, is modified and is optimized, so that gene efficient expression；For example, can be according to recipient plant institute partially The codon of love, change nucleotide sequence while the amino acid sequence of ZmNLP9 genes of the present invention is kept with more preferably suitable Answer the preferences of recipient plant；

2) the gene 5 ' terminal sequence of neighbouring initial methionine is modified, to increase translation efficiency；For example, using in plant Known effective sequence is modified；

3) with the connections of various plant-expressible promoters, to realize the regulation of ZmNLP9 genetic transcription intensity；The startup Son may include composing type, induction type, sequential regulation, growth adjustment, Chemical Regulation, tissue preferably and tissue-specific promoter； The selection of promoter will need and change with expression time and space, and also depend on receptor species requirement；Such as organize Or the specific expressing promoter of organ, acceptor as needed express the ZmNLP9 genes in particular organization or organ；Choosing The expression that dicot promoters are used in dicotyledon is selected, monocotyledonous promoter is used in monocotyledon Expression；

4) it is connected with suitable transcription terminator, to improve the expression efficiency of gene of the present invention；Such as from CaMV's Tml, from rbcS E9；Any known available terminator to be worked in plant can enter with gene of the present invention Row connection；

5) enhancer sequence is introduced, such as intron sequences (such as from Adhl and bronzel) and viral leader sequence (such as from TMV, MCMV and AMV).

And inventor is had found in addition to above-mentioned transcription factor, the expression cassette or core of the nucleic acid molecules comprising the transcription factor Acid vectors also possess same function, wherein：

The nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also be RNA, such as mRNA or hnRNA.

The ZmNLP9 expression casettes refer to express the ZmNLP9's shown in SEQ ID No.2 in host cell DNA, the DNA not only may include the promoter for starting the ZmNLP9 genetic transcriptions, may also include and terminate the ZmNLP9 genes The terminator of transcription.Further, the ZmNLP9 expression casettes may also include enhancer sequence.Startup available for the present invention Son includes but is not limited to：Constitutive promoter, organize, the promoter that organ and development are special, and inducible promoter.Promoter Example include but is not limited to：The constitutive promoter 35S of cauliflower mosaic virus；Wound-inducible from tomato starts Son, and leucine aminopeptidase (" LAP ", Chao et al. (1999) Plant Physiol, 120:979-992)；Change from tobacco Learn inducible promoter (being induced by salicylic acid and BTH (diazosulfide -7- carbothioic acid S-methyl esters))；Tomato protease presses down Formulation II promoter (PIN2) or LAP promoters (available jasmonic acid Yue esters induction)；Heat-shock promoters (United States Patent (USP) 5, 187,267)；Tetracycline inducible promoter (United States Patent (USP) 5,057,422)；Seed specific promoters, as Millet Seed is special Specific Promoters pF128 (CN101063139B (Chinese patent 200710099169.7)), seed storage protein matter is special to be opened Mover (for example, phaseolin, napin, oleosin and soybean beta conglycin promoter (Beachy et al, (1985)EMBO J.4:3047-3053)).All references cited herein is cited in full text.Suitable transcription terminator Including but not limited to：Agrobacterium nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV35S terminators, Tml terminators, pea rbcS E9 terminators and nopaline and octopine synthase terminator (see, e.g.：Odell et al,(1985)Nature,313:810；Rosenberg et al,(1987)Gene,56:125；Guerineau et al, (1991)Molecualr Genomics and Genetics,262:141；Proudfoot(1991)Cell,64:671； Sanfacon et al,Gene and Development,5:141；Mogen et al,(1990)Plant Cell,2: 1261；Munroe et al,(1990)Gene,91:151；Ballad et al,(1989)Nucleic Acids Research,17:7891；Joshi et al,(1987)Nucleic Acids Research,15:9627).

The recombinant expression carrier of the ZmNLP9 expression casettes can be contained with existing plant expression vector construction.It is described Plant expression vector includes double base agrobacterium vector and the carrier available for plant micropellet bombardment etc..As pROKII, pBin438, PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or PCAMBIA1391-Xb (CAMBIA companies) etc..The plant expression vector can also include 3 ' end non-translational regions of foreign gene Domain, i.e., comprising polyadenylation signals and the DNA fragmentation of any other participation mRNA processing or gene expression.The polyadenylic acid letter Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, and such as Agrobacterium crown gall nodule induction (Ti) plasmid gene, (such as kermes closes Into enzyme Nos genes), plant gene (such as soybean storage protein genes) 3 ' end transcription non-translational region be respectively provided with similar functions.Make During with gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used, this A little enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must be with the reading of coded sequence Frame is identical, to ensure the correct translation of whole sequence.The source of the translation control signal and initiation codon be it is extensive, can To be natural or synthesis., can be to institute for the ease of transgenic plant cells or plant are identified and screened It is processed with plant expression vector, the enzyme or light emitting compound of color change can be produced as added the coding that can be expressed in plant The gene (gus gene, luciferase genes etc.) of thing, the marker gene of antibiotic (are such as assigned to kanamycins and related antibiosis The nptII genes of plain resistance, the bar genes to herbicide phosphinothricin resistance are assigned, are assigned to antibiotic hygromycin resistance Hph genes and imparting assign the EPSPS genes to glyphosate to the dhfr genes of methatrexate resistances) or it is anti- Chemical reagent marker gene etc. (such as anti-herbicide gene), the mannose-6-phosphate isomerase base that metabolism mannose ability is provided Cause.

The ZmNLP9 expression vectors can be by using Ti-plasmids, plant virus carrying agent, directly delivered DNA, micro- note Penetrate, the standard biologic technical method such as electroporation import plant cell (Weissbach, 1998, Method forPlantMolecular Biology VIII,Academy Press,NewYork,pp.411-463；Geisersonand Corey,1998,Plant Molecular Biology(2nd Edition)。

The purpose plant can be monocotyledon or dicotyledon.The above method is additionally included in described to recipient plant The plant of screening expression ZmNLP9 genes obtains institute from the plant for importing the ZmNLP9 genes after middle importing ZmNLP9 genes The step of stating genetically modified plants.

The genetically modified plants are interpreted as not only comprising the first generation transgenosis for obtaining the genetic transformation purpose plant Plant, also including its filial generation.For genetically modified plants, the gene can be bred in the species, it is also possible to traditional breeding method The gene transfer is entered to other kinds of same species, particularly including in commercial variety.The genetically modified plants include seed, Callus, intact plant and cell.

Based on it, import the recombinant microorganism of above-mentioned ZmNLP9 expression casettes or import ZmNLP9 bases described above Because the recombinant microorganism of expression vector falls within protection scope of the present invention：

Wherein, the recombinant microorganism concretely bacterium, yeast, algae and fungi.Wherein, bacterium may be from Escherichia Belong to (Escherichia), Erwinia (Erwinia), Agrobacterium tumefaciems category (Agrobacterium), Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), pseudomonas (Pseudomonas), Bacillus (Bacillus) etc..

In summary, after being transferred to arabidopsis using transcription factor provided by the present invention and its derived product, intend with acceptor Southern mustard is compared, and its fresh weight substantially increases, vivo acid content and total nitrogen content increase, main root length dramatically increase.

Brief description of the drawings

Fig. 1 be expression ZmNLP9 genes transgenic Arabidopsis plants RT-PCR Molecular results, wherein WT:It is wild Type, nlp7-4:Mutant, C₉1-8, C₉ 11-9,C₉12-5:The transgenosis that ZmNLP9 sequences are gone in nlp7-4 mutant Strain, experimental result can be seen that ZmNLP9 can be expressed in transgenic line；

Fig. 2 is in 0.2mM, 2.5mM, 5mM KNO₃Under the conditions of turn the transgenic arabidopsis root growths of ZmNLP9 genes Photo, wherein WT:Wild type, nlp7-4:Mutant, C1：ZmNLP9/nlp7-4-1 transgenic lines, C12：ZmNLP9/ Nlp7-4-8 transgenic lines, experimental result can be seen that transgenic line root main root length is longer than adjoining tree；

Fig. 3 is in 0.2mM, 2.5mM, 5mM KNO₃Under the conditions of turn ZmNLP9 genes transgenic arabidopsis root system master Root long degree and fresh weight statistical result, wherein WT:Wild type, nlp7-4:Mutant, ZmNLP9/nlp7-4-1 and ZmNLP9/ Nlp7-4-12 is the transgenic arabidopsis transgenic line for turning ZmNLP9 genes, and experimental result can be seen that transgenic line Main root length and fresh weight are apparently higher than wild type；

Fig. 4 is the transgenic arabidopsis vivo acid content and total nitrogen for turning ZmNLP9 genes in 7 days in 1/2MS Liquid Cultures Assay result, wherein WT:Wild type, nlp7-4:Mutant, C1：ZmNLP9/nlp7-4-1 transgenic lines, C12： ZmNLP9/nlp7-4-12 transgenic lines, experimental result can be seen that amino acid content and total nitrogen content can in transgenic line To recover to wild-type levels.

Embodiment

Further definition is of the invention in following examples, description and these embodiments more than, people in the art Member can determine the essential characteristic of the present invention, and without departing from the spirit and scope of the invention, can be to the present invention Make various changes and modifications, so that it is applicable various uses and condition.In addition to special indicate, of the present invention is this Field prior art；

In an embodiment of the present invention, the startup of the ZmNLP9 genetic transcriptions is started in the ZmNLP9 expression casettes Son is the constitutive promoter 35S of cauliflower mosaic virus, and the terminator for terminating the ZmNLP9 genetic transcriptions is NOS.

In an embodiment of the present invention, hygromycin B of the selectable marker gene for imparting to antibiotic hygromycin resistance Phosphotransferase (hph) gene hyg.The ZmNLP9 genes pass through the ZmNLP9 genes containing the ZmNLP9 expression casettes Expression vector imports purpose plant.The ZmNLP expression vectors are in the insertion of pCAMBIA1300 SalI and KpnI sites The recombinant expression carrier that ZmNLP9 coded sequences shown in SEQ ID No.1 obtain.

The transcription factor ZmNLP9 of embodiment 1 acquisition

Take tri-leaf period Leaves of Maize Seedlings extraction RNA and by its reverse transcription into cDNA.Using cDNA as template, use

Sense primer：5 '-GGGTCGACGCAGCAGCAAGGTTTCATCCCAT ', its nucleotide sequence such as SEQ ID Shown in NO.3；

Anti-sense primer：5 '-GGGGTACCTCAACCAGAGCTTCCACAAGAACTGC-3 ', its nucleotide sequence such as SEQ Shown in ID NO.4；

ZmNLP9 nucleotide sequence is expanded using PCR (PCR), amplification program is：98 DEG C of pre- changes Property 2min；98 DEG C of denaturation 10s, 60 DEG C of annealing 20s, 72 DEG C of extension 2min, 35 circulate；72 DEG C extend 8min eventually.After PCR terminates Enter row agarose gel electrophoresis analysis, amplified fragments obtain the target gene fragment shown in SEQ ID NO.1；Its amino encoded Acid sequence is as shown in SEQ ID NO.2.

The structure of embodiment 2 overexpression arabidopsis carrier and its application

Using pBI121 carriers as template, using primer 35S_F (5 '-AAGCTTatggtggagcacgacactctcga-3 ', Its nucleotide sequence is as shown in SEQ ID NO.5；) and 35S_R：(5’-AAGCTTagagatagatttgtagagagagactgg- 3 ', its nucleotide sequence is as shown in SEQ ID NO.6；), with Pfu high-fidelity enzymatic amplification 35S promoter sequences, PCR reaction bars Part：98 DEG C of pre-degeneration 4min；Then 98 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min, 25 circulations；72 DEG C extend 8min eventually；Due to HindIII restriction enzyme sites are added in primer 35S_F and 35S_R, pcr amplification product is connected into same warp after HindIII digestions Plant expression vector pCAMBIA1300 after HindIII digestions, is connected through digestion and sequencing identification 35S promoter with correct direction The clone entered, it is built into the plant over-express vector containing 35S promoter；Again using pBI121 carriers as template, primer Ter_F is utilized (5 '-GAATTCgatctgtcgatcgacaagctcga-3 ', its nucleotide sequence is as shown in SEQID NO.7；) and Ter_R： (5 '-GAATTCtaattcgggggatctggatttta-3 ', its nucleotide sequence is as shown in SEQ ID NO.8；), with Pfu height Fidelity enzymatic amplification Ter terminator sequences, PCR reaction conditions：98 DEG C of pre-degenerations 4 minutes；Then 98 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds, 25 circulations；Due to adding EcoRI restriction enzyme sites, pcr amplification product warp in primer Ter_F and Ter_R After EcoRI digestions, the new plant expression vector for the firm completion 35S promoter insertion being connected into equally after EcoRI digestions PCAMBIA1300, the clone being connected into through digestion and sequencing identification Ter terminator sequences with correct direction, it is built into and starts containing 35S The plant over-express vector pCAMBIA1300-35S-Ter of son and Ter terminators.

The product for expanding to obtain in embodiment 1 is cut into glue, glue reclaim is carried out with the glue reclaim kit of Omiga companies.Will The fragment being recovered to restriction enzyme SalI and KpnI digestion, and with the pCAMBIA1300- with SalI and KpnI digestions 35S-Ter carriers are attached by T4 ligases, March I competent escherichia coli cells are then converted, by the sun of acquisition Property monoclonal send sequencing, after sequence alignment is correct, upgrading grain.

Between ZmNLP9 ORF fragments are connected in into SalI the and KpnI sites of carrier, structure overexpression arabidopsis carrier.Carry Body has Plant Transformation selection markers hygromycin gene.ZmNLP9ORF fragments on carrier are transferred to Agrobacterium after being sequenced correctly GV3101, infect arabidopsis nlp7-4 mutant with inflorescence infestation method.T0 for seed by 70% ethanol room temperature 5min, 26% time Sodium chlorate room temperature 10min is sterilized, and after sterile water wash 5 times, is uniformly coated on MS flat boards and (is contained hygromycin 25mg/L).4℃ Processing is transferred to 22 DEG C of incubator growths after 3 days.Due to carrying the resistance of hygromycin in plant expression vector pCAMBIA1300 Gene, it can be transferred to plant in conversion with target gene, therefore in hygromycin culture medium, sprout about 7-10 days Afterwards, the leaf of transfer-gen plant is dark green, and root is longer；Non-transformed Plant Leaf is light green, and root is short, it is impossible to long-term surviving.Transformant is moved Enter and grown in Nutrition Soil until receiving T1 for seed.T1 is screened as stated above for seed, and receiving T2, (each strain is received multiple for seed Individual plant).T2 for heterozygote transgenic line due to during filial generation is produced can producer separation and independent assortment, therefore The seed for some amphidromic chloramphenicol resistance occurs；Only T2 can be all containing tide on behalf of the seed of homozygote transgenic line Survived in the MS culture mediums of chloramphenicol resistance.Each T2 is screened respectively for single-strain seed, is planted, and collects the T3 generation kinds of each individual plant Son, the T3 of each individual plant is screened for seed, the unseparated progress RT-PCR identifications (Fig. 1) (resistant to hygromycin) of offspring.

The functional verification of embodiment 3

The strain that will be cultivated based on the seed of the genetically modified plants for being transferred to ZmNLP9 genes obtained in embodiment 2 In KNO₃Concentration is 0.2mM, and the phenotype grown on 2.5mM, 5mM MS vegetable solid culture mediums is analyzed.(the figure from result 2) as can be seen that the main root length and fresh weight of the transgenic Arabidopsis plants of expression ZmNLP9 genes significantly increase compared with recipient plant Add；Under 1/2MS condition of culture, express in the transgenic Arabidopsis plants of ZmNLP9 genes amino acid content and total nitrogen content compared with by Body plant all dramatically increases.

Sequence table

<110>Shandong Agricultural University

<120>One from transcription factor ZmNLP9 of corn and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2781

<212> DNA

<213>Corn (Zea mays)

<400> 1

atggacttcg acccctcccc cgcctccgcc cacggggacg gcagcggcgg cgcctggccc 60

ttcgactcgc tcaccacgtc catgctcttc tcctccgtct ccgcctcccc gcagctgccg 120

gccagctcct cgtcctggct caccccgccc tcgccgctct ggctcttcga ggaccgccac 180

ctgctcccgc tcgacgcccc ctccgccgcg ccggaggcag ccgtcgcagc cgccgtcgtc 240

gaggaggtcc agcgggcacg ctccgggaat tctcacacta caggcaagag agttgatcaa 300

atcaatcaca agtggcagtt tcatctatct ctggacgaag atggcacaga taactctagc 360

ttcttcaagg agaagcttac acaagccctt cggtacttca aggagtccac agatcaacat 420

ctgttggtcc aagtctgggc accagtcaag aacggagacc gctatgtgct tactacttca 480

ggacagcctt ttgtgcttga ccaccagagc attggcctac ttcagtacag ggctgtatcc 540

atgatgtaca tgttttcagt tgatggggaa aatgctggag acctgggcct acctgggcgt 600

gtctacaagc aaaaggtgcc tgagtggaca ccaaatgtgc agtactatag cagtggtgag 660

tatcaacgac ttaaccatgc gatcagttac aatgtccatg gtacggttgc cctgcctgtt 720

tttgatcctt ctgctcagtc gtgtattgct gtcgtcgaac ttataatgac atcaaagaag 780

ataaactatg cctgtgaggt cgataaagtc tgcaaagcac ttgaggcggt aaatcttaaa 840

agcactgaaa tactggacca tccaaatgtc cagatatgta atgaaggccg tcaagcggct 900

ctggtcgaaa tactagagat cttgactgta gtatgtgaag agcacaagct tcctttggca 960

caaacctggg ttccttgcaa gtatagaagt gtattggcac atggtggtgg tttaaagaag 1020

agttgcttaa gtttcgatgg aagttgcatg ggggaagtct gcatgtcaac tagtgatgtg 1080

gcgtttcatg tgattgatgc tcatatgtgg ggatttagag atgcatgcgt ggagcatcac 1140

ctacagaggg gacaaggagt ttctggaaag gcatttatct cacacaaacc ttgcttttca 1200

aaagatatcc gcaagttctg taagttggca tatcctcttg tacactatgc tcgcatgttt 1260

gggttgtctg gctgctttgc aatatgcttg caaagttctt ataccgggaa tgacgactac 1320

atattagagt tctttttgcc actcgattgt attgacgaag atgaccaaaa tgccttactg 1380

gagtctatat taactctgat gaagcggtgt cttcgtagcc taaagctagt tgatgacaat 1440

gatttgagtg gggtttctct tcaccttagt aatgtgctaa aacttgagaa tgaagaatcc 1500

aaaacagatg cacagtttga tggttccctt cgcgaatcac ctgaggatga tagacatgga 1560

ggatcccaca agtttgataa tgagaaccag aaagttttgg atatcacaga gggacaattg 1620

ttgactgatg actactctca agacaatggt acttctgttg gcagaccaaa tggtagtggt 1680

gcttctgatt cttcattgct tcacaaaacc aacaaacccc ctgaaagaag acgtgggaaa 1740

gctgaaaaga ctatcagttt agaggttctt cagcaatatt tttctggaag cttgaaaaac 1800

gcagctaaaa gccttggcgt ttgtcctaca accatgaaac gcatctgccg gcagcatgga 1860

atttctcgtt ggccatctcg gaaaattaat aaggtcaaca ggtctctctc gaaattaaag 1920

caggtgatcg agtctgttca aggctccgat gcggcattta atctcacatc tatcacaggt 1980

cctcttccta tacctgttgg cccttcacca gagtctctca atgtagagaa attaactcaa 2040

agtaaagtgg cagaactttc gaatcttgct gttggtgttg acagagattc tttgcagaag 2100

tcgcttgaaa atgatgacca ttttgatata gtcatggccc agcaaggatt tatagacaat 2160

aacaatgacg tgcagcttga agctgacaaa gcttctcact cgagaagttc atctggagag 2220

ggcagcataa attcacgcac ttcagaaggc tcatgccagg gaagtccagc aaaccaaaca 2280

tttgtttgta agccaatcgc ttcaatgttc gcagaacctc aacttaatca agaggagttc 2340

aacaaagaac cttttcaaga accacaacta ccactttcaa ggatgctgat tgaagattct 2400

ggtagctcca aagatttgaa gaatctcttc acttcaacat cggaccaacc atttttggcc 2460

cctcccaaca acttggtgtc aatgaagcat tcagggactg taacgataaa ggcgagtttt 2520

aaggaagata ttgtgaggtt ccgtttccca tgctcaggtg gtgttattgt attgaaagag 2580

gaggtagcga agaggctaag gatggatgtt ggcacgttcg atatcaagta tctcgatgat 2640

gaccatgagt gggtaaagtt ggcgtgcaat gcagacttgg aagaatgcat ggagatctcg 2700

cggctttctg gcagccatgt cattaggcta ctggttagcg atattgcagc acactttggc 2760

agttcttgtg gaagctctgg t 2781

<210> 2

<211> 927

<212> PRT

<213>Corn (Zea mays)

<400> 2

Met Asp Phe Asp Pro Ser Pro Ala Ser Ala His Gly Asp Gly Ser Gly

1 5 10 15

Gly Ala Trp Pro Phe Asp Ser Leu Thr Thr Ser Met Leu Phe Ser Ser

20 25 30

Val Ser Ala Ser Pro Gln Leu Pro Ala Ser Ser Ser Ser Trp Leu Thr

35 40 45

Pro Pro Ser Pro Leu Trp Leu Phe Glu Asp Arg His Leu Leu Pro Leu

50 55 60

Asp Ala Pro Ser Ala Ala Pro Glu Ala Ala Val Ala Ala Ala Val Val

65 70 75 80

Glu Glu Val Gln Arg Ala Arg Ser Gly Asn Ser His Thr Thr Gly Lys

85 90 95

Arg Val Asp Gln Ile Asn His Lys Trp Gln Phe His Leu Ser Leu Asp

100 105 110

Glu Asp Gly Thr Asp Asn Ser Ser Phe Phe Lys Glu Lys Leu Thr Gln

115 120 125

Ala Leu Arg Tyr Phe Lys Glu Ser Thr Asp Gln His Leu Leu Val Gln

130 135 140

Val Trp Ala Pro Val Lys Asn Gly Asp Arg Tyr Val Leu Thr Thr Ser

145 150 155 160

Gly Gln Pro Phe Val Leu Asp His Gln Ser Ile Gly Leu Leu Gln Tyr

165 170 175

Arg Ala Val Ser Met Met Tyr Met Phe Ser Val Asp Gly Glu Asn Ala

180 185 190

Gly Asp Leu Gly Leu Pro Gly Arg Val Tyr Lys Gln Lys Val Pro Glu

195 200 205

Trp Thr Pro Asn Val Gln Tyr Tyr Ser Ser Gly Glu Tyr Gln Arg Leu

210 215 220

Asn His Ala Ile Ser Tyr Asn Val His Gly Thr Val Ala Leu Pro Val

225 230 235 240

Phe Asp Pro Ser Ala Gln Ser Cys Ile Ala Val Val Glu Leu Ile Met

245 250 255

Thr Ser Lys Lys Ile Asn Tyr Ala Cys Glu Val Asp Lys Val Cys Lys

260 265 270

Ala Leu Glu Ala Val Asn Leu Lys Ser Thr Glu Ile Leu Asp His Pro

275 280 285

Asn Val Gln Ile Cys Asn Glu Gly Arg Gln Ala Ala Leu Val Glu Ile

290 295 300

Leu Glu Ile Leu Thr Val Val Cys Glu Glu His Lys Leu Pro Leu Ala

305 310 315 320

Gln Thr Trp Val Pro Cys Lys Tyr Arg Ser Val Leu Ala His Gly Gly

325 330 335

Gly Leu Lys Lys Ser Cys Leu Ser Phe Asp Gly Ser Cys Met Gly Glu

340 345 350

Val Cys Met Ser Thr Ser Asp Val Ala Phe His Val Ile Asp Ala His

355 360 365

Met Trp Gly Phe Arg Asp Ala Cys Val Glu His His Leu Gln Arg Gly

370 375 380

Gln Gly Val Ser Gly Lys Ala Phe Ile Ser His Lys Pro Cys Phe Ser

385 390 395 400

Lys Asp Ile Arg Lys Phe Cys Lys Leu Ala Tyr Pro Leu Val His Tyr

405 410 415

Ala Arg Met Phe Gly Leu Ser Gly Cys Phe Ala Ile Cys Leu Gln Ser

420 425 430

Ser Tyr Thr Gly Asn Asp Asp Tyr Ile Leu Glu Phe Phe Leu Pro Leu

435 440 445

Asp Cys Ile Asp Glu Asp Asp Gln Asn Ala Leu Leu Glu Ser Ile Leu

450 455 460

Thr Leu Met Lys Arg Cys Leu Arg Ser Leu Lys Leu Val Asp Asp Asn

465 470 475 480

Asp Leu Ser Gly Val Ser Leu His Leu Ser Asn Val Leu Lys Leu Glu

485 490 495

Asn Glu Glu Ser Lys Thr Asp Ala Gln Phe Asp Gly Ser Leu Arg Glu

500 505 510

Ser Pro Glu Asp Asp Arg His Gly Gly Ser His Lys Phe Asp Asn Glu

515 520 525

Asn Gln Lys Val Leu Asp Ile Thr Glu Gly Gln Leu Leu Thr Asp Asp

530 535 540

Tyr Ser Gln Asp Asn Gly Thr Ser Val Gly Arg Pro Asn Gly Ser Gly

545 550 555 560

Ala Ser Asp Ser Ser Leu Leu His Lys Thr Asn Lys Pro Pro Glu Arg

565 570 575

Arg Arg Gly Lys Ala Glu Lys Thr Ile Ser Leu Glu Val Leu Gln Gln

580 585 590

Tyr Phe Ser Gly Ser Leu Lys Asn Ala Ala Lys Ser Leu Gly Val Cys

595 600 605

Pro Thr Thr Met Lys Arg Ile Cys Arg Gln His Gly Ile Ser Arg Trp

610 615 620

Pro Ser Arg Lys Ile Asn Lys Val Asn Arg Ser Leu Ser Lys Leu Lys

625 630 635 640

Gln Val Ile Glu Ser Val Gln Gly Ser Asp Ala Ala Phe Asn Leu Thr

645 650 655

Ser Ile Thr Gly Pro Leu Pro Ile Pro Val Gly Pro Ser Pro Glu Ser

660 665 670

Leu Asn Val Glu Lys Leu Thr Gln Ser Lys Val Ala Glu Leu Ser Asn

675 680 685

Leu Ala Val Gly Val Asp Arg Asp Ser Leu Gln Lys Ser Leu Glu Asn

690 695 700

Asp Asp His Phe Asp Ile Val Met Ala Gln Gln Gly Phe Ile Asp Asn

705 710 715 720

Asn Asn Asp Val Gln Leu Glu Ala Asp Lys Ala Ser His Ser Arg Ser

725 730 735

Ser Ser Gly Glu Gly Ser Ile Asn Ser Arg Thr Ser Glu Gly Ser Cys

740 745 750

Gln Gly Ser Pro Ala Asn Gln Thr Phe Val Cys Lys Pro Ile Ala Ser

755 760 765

Met Phe Ala Glu Pro Gln Leu Asn Gln Glu Glu Phe Asn Lys Glu Pro

770 775 780

Phe Gln Glu Pro Gln Leu Pro Leu Ser Arg Met Leu Ile Glu Asp Ser

785 790 795 800

Gly Ser Ser Lys Asp Leu Lys Asn Leu Phe Thr Ser Thr Ser Asp Gln

805 810 815

Pro Phe Leu Ala Pro Pro Asn Asn Leu Val Ser Met Lys His Ser Gly

820 825 830

Thr Val Thr Ile Lys Ala Ser Phe Lys Glu Asp Ile Val Arg Phe Arg

835 840 845

Phe Pro Cys Ser Gly Gly Val Ile Val Leu Lys Glu Glu Val Ala Lys

850 855 860

Arg Leu Arg Met Asp Val Gly Thr Phe Asp Ile Lys Tyr Leu Asp Asp

865 870 875 880

Asp His Glu Trp Val Lys Leu Ala Cys Asn Ala Asp Leu Glu Glu Cys

885 890 895

Met Glu Ile Ser Arg Leu Ser Gly Ser His Val Ile Arg Leu Leu Val

900 905 910

Ser Asp Ile Ala Ala His Phe Gly Ser Ser Cys Gly Ser Ser Gly

915 920 925

<210> 3

<211> 31

<212> DNA

<213>Artificial sequence ()

<400> 3

gggtcgacgc agcagcaagg tttcatccca t 31

<210> 4

<211> 34

<212> DNA

<213>Artificial sequence ()

<400> 4

ggggtacctc aaccagagct tccacaagaa ctgc 34

<210> 5

<211> 29

<212> DNA

<213>Artificial sequence ()

<400> 5

aagcttatgg tggagcacga cactctcga 29

<210> 6

<211> 33

<212> DNA

<213>Artificial sequence ()

<400> 6

aagcttagag atagatttgt agagagagac tgg 33

<210> 7

<211> 29

<212> DNA

<213>Artificial sequence ()

<400> 7

gaattcgatc tgtcgatcga caagctcga 29

<210> 8

<211> 29

<212> DNA

<213>Artificial sequence ()

<400> 8

gaattctaat tcgggggatc tggatttta 29

Claims (3)

1. A 1. transcription factor ZmNLP9 for deriving from corn, it is characterised in that：The nucleotide sequence of the transcription factor such as SEQ Shown in ID NO.1, the amino acid sequence that it is encoded is as shown in SEQ ID NO.2.
2. 2. it is overexpressed purposes of the transcription factor ZmNLP9 on plant nitrogen utilization ratio is improved.
3. 3. purposes according to claim 2, it is characterised in that：

   The purposes of expression cassette or nucleic acid carrier comprising transcription factor ZmNLP9 on plant nitrogen utilization ratio is improved.