CN107602683A - One from transcription factor ZmNLP4 of corn and application thereof - Google Patents

Abstract

The present invention relates to field of plant genetic, one is provided from transcription factor ZmNLP4 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, its total biomass substantially increases, vivo acid content and total nitrogen content increase, main root length and lateral root number substantially increase, single plant yield dramatically increases, nitrate transport protein gene expression amount, Nitrite reductase expression quantity and nitrite reductase gene expression quantity significantly raise.

Description

One from transcription factor ZmNLP4 of corn and application thereof

Technical field

The present invention relates to field of plant genetic, there is provided one from corn transcription factor ZmNLP4 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 ZmNLP4 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 total biomass substantially increases the arabidopsis after being transferred to using the transcription factor Add, vivo acid content and total nitrogen content increase, main root length and lateral root number substantially increases, single plant yield dramatically increases, Nitrate transport protein gene expression amount, Nitrite reductase expression quantity and nitrite reductase gene expression quantity are notable Rise.

This concrete technical scheme of the invention is as follows：

One transcription factor ZmNLP4 for deriving from corn, the nucleotide sequence such as SEQ ID NO.1 institutes of the transcription factor Showing, for the amino acid sequence that it is encoded as shown in SEQ ID NO.2, above-mentioned transcription factor ZmNLP4 is inventor by PCR method, Using tri-leaf period Leaves of Maize Seedlings extract RNA and by its reverse transcription into cDNA as template, utilize following primer amplification obtain：

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

Anti-sense primer：5 '-ggtaccACCGGAGCTTCCACAAGAACTGCCA-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 ZmNLP4 genes：

With nitrate nitrogen (NO₃ ^-) be only nitrogen source under conditions of, be transferred to the arabidopsis of ZmNLP4 genes and acceptor arabidopsis Compare, its total biomass substantially increases, vivo acid content and total nitrogen content increase, main root length and lateral root number substantially increase Add, single plant yield dramatically increases, nitrate transport protein gene expression amount, Nitrite reductase expression quantity and nitrite Reductase gene expression quantity significantly raises.

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；

D, lateral root number is more than the recipient plant；

E, plant single plant yield is higher than the recipient plant.

Based on above-mentioned transcription factor ZmNLP4, 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 ZmNLP4 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 ZmNLP4 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 ZmNLP4 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 ZmNLP4 expression casettes refer to express the ZmNLP4's shown in SEQ ID No.2 in host cell DNA, the DNA not only may include the promoter for starting the ZmNLP4 genetic transcriptions, may also include and terminate the ZmNLP4 genes The terminator of transcription.Further, the ZmNLP4 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 ZmNLP4 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 ZmNLP4 expression vectors can be by using Ti-plasmids, plant virus carrying agent, directly delivered DNA, microinjection, electricity The standard biologic technical methods such as perforation 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 ZmNLP4 genes obtains institute from the plant for importing the ZmNLP4 genes after middle importing ZmNLP4 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 ZmNLP4 expression casettes or import ZmNLP4 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 total biomass substantially increases, vivo acid content and total nitrogen content increase, main root length and lateral root number are bright Aobvious increase, single plant yield dramatically increase, nitrate transport protein gene expression amount, Nitrite reductase expression quantity and nitrous Hydrochlorate reductase gene expression quantity significantly raises.

Brief description of the drawings

Fig. 1 is the RT-PCR Molecular results of the transgenic Arabidopsis plants of expression ZmNLP4 genes,

Wherein WT:Wild type, nlp7-4:Mutant, ZmNLP4/nlp7-4-8-1, ZmNLP4/nlp7-4-8, ZmNLP4/ nlp7-4-12,ZmNLP4/nlp7-4-14:The transgenic line that ZmNLP4 sequences are gone in nlp7-4 mutant, experiment knot Fruit can be seen that ZmNLP4 can be expressed in transgenic line；

Fig. 2 is in 0.2mM, 2.5mM, 5mM KNO₃Under the conditions of turn ZmNLP4 genes transgenic arabidopsis grow 10 days Picture, wherein WT:Wild type, nlp7-4:Mutant, C1：ZmNLP4/nlp7-4-1 transgenic lines, C8：ZmNLP4/ Nlp7-4-8 transgenic lines, experimental result can be seen that the transgenic line growth for turning ZmNLP4 nucleotide sequences significantly better than open country Raw type and mutant；

Fig. 3 is in 0.2mM, 2.5mM, 5mM KNO₃Under the conditions of turn ZmNLP4 genes transgenic arabidopsis grow 10 days Biomass statistics result, wherein WT:Wild type, nlp7-4:Mutant, ZmNLP4/nlp7-4-1, ZmNLP4/nlp7-4-8, ZmNLP4/nlp7-4-12,ZmNLP4/nlp7-4-14:Transgenic line, experimental result can be seen that the fresh weight of transgenic line With dry weight apparently higher than wild type；

Fig. 4 is the transgenic arabidopsis vivo acid content and total nitrogen for turning ZmNLP4 genes in 7 days in 1/2MS Liquid Cultures Assay result, wherein WT:Wild type, nlp7-4:Mutant, C1：ZmNLP4/nlp7-4-1 transgenic lines, C8： ZmNLP4/nlp7-4-8 transgenic lines, C12：ZmNLP4/nlp7-4-12 transgenic lines, C14：ZmNLP4/nlp7-4-14 Transgenic line, experimental result can be seen that amino acid content and total nitrogen content can recover to wild type water in transgenic line It is flat；

Fig. 5 is in 0.2mM, 2.5mM, 5mM KNO₃Under the conditions of turn the transgenic arabidopsis root growths of ZmNLP4 genes Photo, wherein WT:Wild type, nlp7-4:Mutant, C1：ZmNLP4/nlp7-4-1 transgenic lines, C8：ZmNLP4/nlp7- 4-8 transgenic lines, experimental result can be seen that the growth of transgenic line underground part can return to wild-type levels；

Fig. 6 is in 0.2mM, 2.5mM, 5mM KNO₃Under the conditions of turn ZmNLP4 genes transgenic arabidopsis root system master Root long degree and lateral root number statistical result, wherein WT:Wild type, nlp7-4:Mutant, C1：ZmNLP4/nlp7-4-1 transgenosis Strain, C8：ZmNLP4/nlp7-4-8 transgenic lines, C12：ZmNLP4/nlp7-4-12 transgenic lines, C14：ZmNLP4/ Nlp7-4-14 transgenic lines, experimental result can be seen that transgenic line main root length and lateral root number apparently higher than open country Raw type；

Fig. 7 is in 0.2mMKNO₃Under the conditions of turn ZmNLP4 genes transgenic arabidopsis single plant yield statistical result, its Middle WT:Wild type, nlp7-4:Mutant, C1：ZmNLP4/nlp7-4-1 transgenic lines, C8：ZmNLP4/nlp7-4-8 turns base Because of strain, experimental result can be seen that under the conditions of low nitrogen transgenic line single plant yield apparently higher than wild type；

Fig. 8 is that the transgenic arabidopsis nitrate for turning ZmNLP4 genes absorbs the expression of marker gene, wherein：

A is the expression of nitrate transport protein gene NRT2.1 after nitric acid Salt treatment,

B is the expression of nitrate reductase gene NIA1 after nitric acid Salt treatment,

C is the expression of nitrite reductase gene NiR after nitric acid Salt treatment；

Its condition of culture is 2.5mM NH₄- Su grows 7 days, 10mM KNO₃/ KCl is handled 2 hours, WT:Wild type, nlp7-4:Mutant, C1：ZmNLP4/nlp7-4-1 transgenic lines, C8：ZmNLP4/nlp7-4-8 transgenic lines, C12： ZmNLP4/nlp7-4-12 transgenic lines, C14：ZmNLP4/nlp7-4-14 transgenic lines, experimental result, which can be seen that, to be turned Nitrate responsive genes NIA1, NiR, NRT2.1 expression can recover to wild-type levels in gene strain.

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 ZmNLP4 genetic transcriptions is started in the ZmNLP4 expression casettes Son is the constitutive promoter 35S of cauliflower mosaic virus, and the terminator for terminating the ZmNLP4 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 ZmNLP4 genes pass through the ZmNLP4 genes containing the ZmNLP4 expression casettes Expression vector imports purpose plant.The ZmNLP4 expression vectors are inserted in pCAMBIA1300 PstI and BamHI sites Enter the recombinant expression carrier that the ZmNLP4 coded sequences shown in SEQ ID No.1 obtain.

The transcription factor ZmNLP4 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 '-ctgcagGTACGATCACACTAGCACCGCCAGC-3 ', its nucleotide sequence such as SEQ ID Shown in NO.3；

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

ZmNLP4 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 2min, 72 DEG C of extension 2min, 35 circulate；72 DEG C extension 8min, PCR terminate eventually Laggard row agarose gel electrophoresis analysis, amplified fragments obtain the target gene fragment shown in SEQ ID NO.1；Its ammonia encoded Base 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 SEQ ID 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 PstI and BamHI digestion, and with the pCAMBIA1300- with PstI and BamHI 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 ZmNLP4 ORF fragments are connected in into PstI the and BamHI sites of carrier, structure overexpression arabidopsis carrier. Carrier has Plant Transformation selection markers hygromycin gene.ZmNLP4ORF 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, (result is such as the unseparated progress RT-PCR identifications (resistant to hygromycin) of offspring Shown in Fig. 1).

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 ZmNLP4 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- Fig. 7) as can be seen that the growing way of the transgenic Arabidopsis plants of expression ZmNLP4 genes is significantly better than recipient plant, express The biomass (including fresh weight and dry weight) of the transgenic Arabidopsis plants of ZmNLP4 genes dramatically increases compared with recipient plant；1/2MS Under condition of culture, amino acid content and total nitrogen content are expressed in the transgenic Arabidopsis plants of ZmNLP4 genes compared with recipient plant all Dramatically increase；And the main root length and lateral root number of the transgenic Arabidopsis plants of expression ZmNLP4 genes are also significantly more than acceptor and planted Thing.Under the conditions of low nitrogen, the single plant yield for expressing the transgenic Arabidopsis plants of ZmNLP4 genes is obvious compared with recipient plant Increase.Fig. 8 is shown, by 10mM KNO₃After induction, Nitrate-N Use gene NRT2.1, NIA1 and NiR is induced table by nitrate nitrogen Up to significantly increasing.

Sequence table

<110>Shandong Agricultural University

<120>One from transcription factor ZmNLP4 of corn and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2748

<212> DNA

<213>Corn (Zea mays)

<400> 1

atggacctcg acccggccta tcccccatca gcctgcgccg acgcctggcc ctttgactcg 60

ctcaccacgt ccctgctctt ctccccgcag ctcacgccga taccggccaa ctcatccttc 120

tcctggctca ccccacactc gccgctctgg ctcttcgagg accgccacct gctcccgctc 180

gacgcgcccg ccgtcttcga agaggtccag cggccacgct ccgggaattc tcacacttca 240

agcaagccag ctgaacagat cagtcacaga tggcagtttc atctgtccct ggacgaagat 300

ggcacagaca actccagctt gttcaaggag aagctgacac acgctcttcg ctacttcaag 360

gagtccacag atcaacatct gctggtccaa gtctgggcac cagtcaagaa tggagaccgc 420

tatgtgctca ctacttcagg acagcctttc gtgcttgacc accagagcat tggcctactt 480

cagtacaggg ctgtatccat gacctacatg ttttcagttg atgcggaaaa tgttggagac 540

ctgggcctac ctgggcgtgt ctataagcaa aaggtgcccg agtggacacc gaatgtgcag 600

tactatagca gtggtgagta ccaacgactt aaccatgcga tcagctacaa cgtccatggt 660

acagtcgccc tgcctgtttt tgatccttcc gctcactcgt gtatcgctgt tgttgaactt 720

ataatgacat caaagaagat aaactatgcc tgcgaggtcg ataaagtctg caaagcactt 780

gaggcggtaa atcttaaaag cactgaaata catgaccatc caaatgtcca gatatgcaat 840

gaaggccgtc aagctgcgct ggtcgaaata ctggagatct tgaccgtagt atgtgaagag 900

cacaagcttc ctttggcaca gacctgggtt ccttgcaagt atagaagtgt cttggcgcat 960

ggtggtggtt taaaaaagag ctgcttaagt tttgatggaa gttgcatggg ggaggtctgc 1020

atgtcaacta gtgatgtggc gtttcatgtg attgatgctc atatgtgggg gtttagagat 1080

gcctgtgtcg agcatcacct acagagggga caaggagttc ctggaaaggc gtttatctca 1140

cacaaacctt gcttttcaaa agatatccgc aagttctgta agttggcata tccccttgta 1200

cactatgctc gcatgtttgg gttggctggc tgctttgcaa tatgcttgca aagttcttat 1260

actggaaatg atgactacat attagagttt ttcttgccgc ccgattgtag tgacgaagat 1320

gaccaaaatg ccttactgga gtctgtatta actctgatga agaggtgtct tcggagccta 1380

aaggtagttg gtgacagaga tttgagtggg gtctctcttc accttagcaa tgtgctaaaa 1440

cttgagaatg aagaattcaa gacagatgca cagtttgata actctgatgg ttccctttgc 1500

caatcaccta acggtgatag acatggagga tcccatgagt ttgatggtgg gaacaagaag 1560

gttttggatg tgccagaggg acaattgttg actgatgact tctctcaagg caatggtact 1620

tctgctggca gaccaaatgg tagcggtgct tctgattctt ctgtgcttca caaaaccaac 1680

aaaccccctg aaaggaaacg tgggaaagcc gaaaagacta tcagtttaga ggttcttcag 1740

caatattttt ctggaagctt gaagaacgca gctaaaagcc ttggtgtttg tcctacaact 1800

atgaagcgca tttgccggca gcatgggatt tctcgttggc catctcgaaa aatcaataag 1860

gtcaaccgat ctctctcgaa actaaagcag gtaatcgagt ctgttcaagg ctccgatgcg 1920

gcatttaatc tcacatctat cacaggtcct ctccctattc ctgttggccc ttcattggat 1980

tctctcaatg tagagaaatt aactcaaagt aaagtggcag aactttcaaa tatcgctgtt 2040

gatggtgaca gagattcttt gcaaaagtca cttggaaatg atggccattt tggtatagtc 2100

atgccccagc aaggatctac agaccataac aatgacacac agcttgaagc tgacaaggcg 2160

tctcactcga gaagttcgtc tggagaaggc agcataaatt cacgcacttc ggaaggctca 2220

tgccagagaa gtccagcgaa ccagacattt gtttgcaagc caattgcttc aacattcgca 2280

gaacctcaac ttaatccaga ggagttcaat aaagagcctt tccaagaacc acaactacca 2340

ctttccagga tgctcattga agattctggt agctccaaag atttgaagaa tctcttcact 2400

tcaacatctg accaaccatt tttggcccct cccaataatt tggtgccaat gaagcattca 2460

gggtctgtaa cgataaaggc gagttttaaa gaagatattg tgaggttccg tttctcatgc 2520

tcaggcagtg ttattgtatt gaaagaggag gtagcgaaaa ggctaaggat ggatgttggc 2580

acgtttgata tcaagtatct agatgatgac catgaatggg taaagttgac gtgtaattca 2640

gacttggaag aatgcatgga gatctcacgg ctttcgggta gccatgtcat caggttatct 2700

gttagcgata ttgcgacaca ctttggcagt tcttgtggaa gctccggt 2748

<210> 2

<211> 916

<212> PRT

<213>Corn (Zea mays)

<400> 2

Met Asp Leu Asp Pro Ala Tyr Pro Pro Ser Ala Cys Ala Asp Ala Trp

1 5 10 15

Pro Phe Asp Ser Leu Thr Thr Ser Leu Leu Phe Ser Pro Gln Leu Thr

20 25 30

Pro Ile Pro Ala Asn Ser Ser Phe Ser Trp Leu Thr Pro His Ser Pro

35 40 45

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

50 55 60

Val Phe Glu Glu Val Gln Arg Pro Arg Ser Gly Asn Ser His Thr Ser

65 70 75 80

Ser Lys Pro Ala Glu Gln Ile Ser His Arg Trp Gln Phe His Leu Ser

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gln Tyr Arg Ala Val Ser Met Thr Tyr Met Phe Ser Val Asp Ala Glu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Val Phe Asp Pro Ser Ala His Ser Cys Ile Ala Val Val Glu Leu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

His Tyr Ala Arg Met Phe Gly Leu Ala Gly Cys Phe Ala Ile Cys Leu

405 410 415

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

420 425 430

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

435 440 445

Val Leu Thr Leu Met Lys Arg Cys Leu Arg Ser Leu Lys Val Val Gly

450 455 460

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

465 470 475 480

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

485 490 495

Gly Ser Leu Cys Gln Ser Pro Asn Gly Asp Arg His Gly Gly Ser His

500 505 510

Glu Phe Asp Gly Gly Asn Lys Lys Val Leu Asp Val Pro Glu Gly Gln

515 520 525

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

530 535 540

Pro Asn Gly Ser Gly Ala Ser Asp Ser Ser Val Leu His Lys Thr Asn

545 550 555 560

Lys Pro Pro Glu Arg Lys Arg Gly Lys Ala Glu Lys Thr Ile Ser Leu

565 570 575

Glu Val Leu Gln Gln Tyr Phe Ser Gly Ser Leu Lys Asn Ala Ala Lys

580 585 590

Ser Leu Gly Val Cys Pro Thr Thr Met Lys Arg Ile Cys Arg Gln His

595 600 605

Gly Ile Ser Arg Trp Pro Ser Arg Lys Ile Asn Lys Val Asn Arg Ser

610 615 620

Leu Ser Lys Leu Lys Gln Val Ile Glu Ser Val Gln Gly Ser Asp Ala

625 630 635 640

Ala Phe Asn Leu Thr Ser Ile Thr Gly Pro Leu Pro Ile Pro Val Gly

645 650 655

Pro Ser Leu Asp Ser Leu Asn Val Glu Lys Leu Thr Gln Ser Lys Val

660 665 670

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

675 680 685

Lys Ser Leu Gly Asn Asp Gly His Phe Gly Ile Val Met Pro Gln Gln

690 695 700

Gly Ser Thr Asp His Asn Asn Asp Thr Gln Leu Glu Ala Asp Lys Ala

705 710 715 720

Ser His Ser Arg Ser Ser Ser Gly Glu Gly Ser Ile Asn Ser Arg Thr

725 730 735

Ser Glu Gly Ser Cys Gln Arg Ser Pro Ala Asn Gln Thr Phe Val Cys

740 745 750

Lys Pro Ile Ala Ser Thr Phe Ala Glu Pro Gln Leu Asn Pro Glu Glu

755 760 765

Phe Asn Lys Glu Pro Phe Gln Glu Pro Gln Leu Pro Leu Ser Arg Met

770 775 780

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

785 790 795 800

Ser Thr Ser Asp Gln Pro Phe Leu Ala Pro Pro Asn Asn Leu Val Pro

805 810 815

Met Lys His Ser Gly Ser Val Thr Ile Lys Ala Ser Phe Lys Glu Asp

820 825 830

Ile Val Arg Phe Arg Phe Ser Cys Ser Gly Ser Val Ile Val Leu Lys

835 840 845

Glu Glu Val Ala Lys Arg Leu Arg Met Asp Val Gly Thr Phe Asp Ile

850 855 860

Lys Tyr Leu Asp Asp Asp His Glu Trp Val Lys Leu Thr Cys Asn Ser

865 870 875 880

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

885 890 895

Ile Arg Leu Ser Val Ser Asp Ile Ala Thr His Phe Gly Ser Ser Cys

900 905 910

Gly Ser Ser Gly

915

<210> 3

<211> 31

<212> DNA

<213>Artificial sequence ()

<400> 3

ctgcaggtac gatcacacta gcaccgccag c 31

<210> 4

<211> 34

<212> DNA

<213>Artificial sequence ()

<400> 4

ggtacctcaa ccggagcttc cacaagaact gcca 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 ZmNLP4 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 ZmNLP4 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 ZmNLP4 on plant nitrogen utilization ratio is improved.