CN109988231A - Paddy gene OsGRF4 is improving the application in plant cold tolerance - Google Patents

Abstract

The present invention provides paddy gene OsGRF4 and is improving the application in plant cold tolerance.Paddy gene OsGRF4 of the present invention is panicled characters controlling gene, derives from the big grain in rice variety river, and the raising of the gene expression amount leads to the enhancing of rice cold tolerance.The experimental results showed that, the survival rate that OsGRF4 overexpresses transgenic line under cold stress is significantly higher than wild type, and OsGRF4 inhibits the survival rate of express transgenic strain to be substantially less than wild type, first demonstration that OsGRF4 gene has the function of enhancing rice cold tolerance.OsGRF4 gene has biggish application prospect for cultivating cold-tolerant rice, and the present invention is that plant cold resistance breeding, especially rice cold-tolerance breeding provide new genetic resources.

Description

Paddy gene OsGRF4 is improving the application in plant cold tolerance

Technical field

The invention belongs to plant genetic engineering fields, specifically, it is cold-resistant in raising plant to be related to paddy gene OsGRF4 Application in property.

Background technique

It is announced according to world Realtime Statistics library, in January, 2019, global population has reached 77.15 hundred million.According to annual 100000000 growth trend, to the year two thousand fifty, population in the world sum will be close to 10,000,000,000.It was predicted that the middle of this century, global agriculture Than 2005 raising 60%-120% of industry level can just support this 10,000,000,000 population.Rice is one of most important cereal crops, The whole world has the population of more than half using rice as staple food, the grain security of the Relationship with Yield of paddy to the world.Rice originates from The torrid zone, subtropical zone, are thermophilous, very sensitive to low temperature.Chilling injury seriously restricts the sown area and sowing model of rice It encloses, while seriously affecting the high yield and stable yields of rice.It is annual to cause paddy underproduction 50-100 hundred million public because damaging to plants caused by sudden drop in temperature only by taking China as an example Jin (Zhao Zhengwu etc., southwest agricultural journal, 2006,19:330-335).Therefore, currently there is an urgent need to excavate more cold-resistant functions Gene, the cold-resistant kind of cultivation damage to plants caused by sudden drop in temperature the loss caused by rice yield to reduce.

According to the growth phase of rice, paddy cool injury can be divided into sprouting stage damages to plants caused by sudden drop in temperature, seedling stage damages to plants caused by sudden drop in temperature and damages to plants caused by sudden drop in temperature with reproduction period.Water The cold resistance of rice is mostly controlled by quantitative trait locus, currently, researcher utilizes Xian/round-grained rice (minority is round-grained rice/round-grained rice or open country/cultivation) hereditary group Body carries out linkage analysis or association analysis using natural population, navigate to more cold-resistant QTL (Lv et al., Plant Cell Environ,2016,39(3):556-570；Wang et al.,Rice,2016,9(1):61).However, The cold-resistant QTL of clone is very few, seriously constrains the molecular breeding process of rice cold tolerance.Therefore, resistance to there is an urgent need to accelerate rice More new rice cold tolerance genes are excavated in introduction, initiative and the evaluation of cold resource, are apparent rice cold tolerance molecule mechanism and regulation Network lays the foundation, and also provides genetic resources and technical support faster and better to cultivate the new rice variety of cold resistance enhancing, Guarantee China's grain security is of great significance to.

Summary of the invention

The object of the present invention is to provide paddy gene OsGRF4 to improve the application in plant cold tolerance.

In order to achieve the object of the present invention, in a first aspect, the present invention, which provides paddy gene OsGRF4, is improving plant cold tolerance In application.Wherein, the paddy gene OsGRF4 is Rice Panicle Traits controlling gene, derives from the big grain in rice variety river. OsGRF4 is the following protein (a) of coding or gene (b):

(a) protein that the amino acid sequence shown in SEQ ID NO:3 forms；

(b) sequence shown in SEQ ID NO:3 is substituted, lacks or adds one or several amino acid and has same function The protein as derived from (a).

Optionally, the nucleotide sequence of the paddy gene OsGRF4 includes the choosing from the following group nucleotide sequence (1)-(7) The nucleotide sequence selected:

(1) nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2；

(2) nucleotide sequence with the nucleotide hybridization of (1) and the cold-resistant GAP-associated protein GAP of coded plant under strict conditions；

(3) with the nucleotide sequence of (1) have at least 70%, preferably at least 80%, more preferably at least 90%, especially extremely The nucleotide sequence of few 95% or 98% or 99% identity；

(4) with the protein of the nucleotide sequence coded same amino acid sequence of (1) but the degeneracy because of genetic codon And nucleotide sequence different in sequence；

(5) nucleotide sequence of one of following amino acid sequence: amino acid sequence shown in SEQ ID NO:3 is encoded, or Person, since one or more (such as 1-25,1-20,1-15,1-10,1-5,1-3) amino acid residues replace In generation, lacks and/or is inserted into and the amino acid sequence different from amino acid sequence shown in SEQ ID NO:3, alternatively, with SEQ ID Amino acid sequence shown in NO:3 have at least 70%, preferably at least 80%, more preferably at least 90%, especially at least 95% or The amino acid sequence of 98% identity；

(6) active fragment of (1)-(5) any nucleotide sequence；Or

(7) nucleotide sequence complementary with (1)-(5) any nucleotide sequence.

Wherein, nucleotide sequence defined in (2)-(7) has the function of enhancing plant cold tolerance.

In the present invention, the stringent condition can be for 6 × SSC, the solution of 0.5%SDS hybridizes at 65 DEG C, then with 2 × SSC, 0.1%SDS and 1 × SSC, it is primary that 0.1%SDS respectively washes film.

In the present invention, the plant includes but is not limited to rice, corn, sorghum, wheat and barley.

Second aspect, the present invention provide a kind of method for improving plant cold tolerance, which comprises

1) making plant includes paddy gene OsGRF4；Or

2) plant is made to express paddy gene OsGRF4.

Method above-mentioned, the method includes but be not limited to transgenosis, hybridization, backcrossing, selfing or vegetative propagation.

Further, the method includes utilizing Ti-plasmids, plant viral vector, directly delivered DNA, microinjection, base It is planted because the conventional biology methods such as rifle, conductance, mediated by agriculture bacillus will be imported comprising the recombinant expression carrier of paddy gene OsGRF4 Object cell obtains transgenic plant；Alternatively, the plant containing paddy gene OsGRF4 is hybridized with another plant, hybridized Offspring.Its cold resistance of Progeny plants obtained is improved.

The third aspect, the present invention provide the promoter of paddy gene OsGRF4, the promoter are as follows:

I) nucleotide sequence shown in SEQ ID NO:4；

Ii) nucleotide sequence shown in SEQ ID NO:4 be substituted, lack and/or increase one or more nucleotide and Nucleotide sequence with promoter activity；

Iii) hybridize and have under strict conditions the nucleotides sequence of promoter activity with sequence shown in SEQ ID NO:4 Column；

Iv) and i), ii) or nucleotide sequence iii) have at least 70%, preferably at least 80%, more preferably at least 90%, the nucleotide sequence of especially at least 95% or 98% or 99% identity；

V) i)~iv) any nucleotide sequence active fragment；Or

Vi) the nucleotide sequence complementary with any nucleotide sequence of i)~iv).

The present invention also provides the biomaterials comprising the paddy gene OsGRF4 promoter.

By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that

(1) have the function of enhancing plant cold tolerance, OsGRF4 gene the invention firstly discloses rice Os GRF4 gene It has great significance to the cold-resistant Regulation Mechanism of plant and genetic neural network is furtherd elucidate.

(2) it is wild to show that the survival rate of the OsGRF4 overexpression transgenic paddy rice strain under cold stress is significantly higher than for experiment OsGRF4 gene can be used for genetic modification of plants by type, to obtain the plant of cold resistance enhancing.

(3) present invention is that plant cold resistance breeding, especially rice cold-tolerance breeding provide new genetic resources, to cultivation Cold-resistant New Crop Varieties ensure that crop production has safely important application value.

Detailed description of the invention

Fig. 1 be in the embodiment of the present invention 1 NIL-OsGRF4 and NIL-Osgrf4 respectively at 8 DEG C of low-temperature treatment 6 days cold-resistant Phenotype.Wherein, NIL-S and NIL-L respectively indicate NIL-Osgrf4 and NIL-OsGRF4.

Fig. 2 is overexpression vector building and the hygromycin PCR detection and sequencing of transgenosis single plant in the embodiment of the present invention 2 Analysis.Wherein, A is overexpression vector figure；B is that PCR amplification hygromycin gene detects transgenic positive single plant；C is sequencing analysis Transgenic positive single plant.

Fig. 3 is that wild rice and OsGRF4 gene overexpress strain respectively at 8 DEG C of low-temperature treatments in the embodiment of the present invention 2 6 days cold-resistant phenotypes.Wherein, WT and OX respectively indicates wild type and OsGRF4 overexpresses transgenic line.

Fig. 4 is wild rice in the embodiment of the present invention 3 and OsGRF4 gene interference strain respectively at 8 DEG C of low-temperature treatments 6 It cold-resistant phenotype.Wherein, WT and RNAi respectively indicates wild type and OsGRF4 interferes transgenic line.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

The discovery of 1 paddy gene OsGRF4 of embodiment enhancing plant cold tolerance new function

The present inventor has cloned Rice Panicle Traits controlling gene OsGRF4, http at first: // Rice.plantbiology.msu.edu/ database accession number: LOC_Os02g47280.The present inventor is in subsequent research process In, find that overexpression osa-MIR396c gene can reduce rice to the tolerance of salt and alkaline stress by consulting literatures, A target gene of the OsGRF4 as osa-MIR396c, the rice in the processing of physiological saline, anoxic, abscisic acid and gibberellin The expression quantity of OsGRF4 raises, and in arsenic acid salt treatment the expression quantity of OsGRF4 show as lowering (Gao et al., Planta,2010,231:991–1001).In addition, Kyndt etc. passes through gene differential expression the study found that rice is in root-knot nematode Infect expression quantity there were significant differences (Kyndt et al., the New Phytologist, 2012,196:887-of front and back OsGRF4 900).Therefore, the present inventor speculates that OsGRF4 may be related to plant stress-resistance, and using the big grain in rice varieties river and huge fringe rice as material Material by hybridization, be selfed and backcrossing construct near isogenic lines NIL-OsGRF4 and NIL-Osgrf4 (Sun et al., JIPB, 2016,58:836-847), wherein the OsGRF4 is to be mutated excellent allele, Osgrf4 is function forfeiture type allele. Analysis and research are compared with the cold resistance of NIL-Osgrf4 to NIL-OsGRF4 by cold stress, as the result is shown after cold treatment The survival rate of NIL-OsGRF4 plant is significantly higher than the survival rate of NIL-Osgrf4, increases by about 50% survival rate (Fig. 1), shows Plant containing gene OsGRF4 obviously shows cold resistance.It can be seen that paddy gene OsGRF4 has enhancing plant cold-resistant New function.Above-mentioned cold treatment method are as follows: 50 plants growth 7 days and health rice seedling by 8 DEG C low-temperature treatment 6 days, so Restore normal temperature afterwards and grow 5 days (28 DEG C of daytime, at night 25 DEG C), finally investigates its survival rate, above-mentioned each material is arranged 3 It repeats.

The cDNA sequence of the above-mentioned excellent allele OsGRF4 that can improve cold resistance is as shown in SEQ ID NO:1, base Because group nucleotide sequence is as shown in SEQ ID NO:2, the amino acid sequence of coding protein as shown in SEQ ID NO:3, Promoter nucleotide sequence is as shown in SEQ ID NO:4.

The building of 2 gene OsGRF4 overexpression vector of embodiment and the genetic transformation of rice

The building of overexpression vector: BamH I restriction enzyme site is had according to the design of the genome sequence of target gene OsGRF4 Primer GRF-OX (F:5 '-gcaggtcgacggatcctactacgccacgagataacgaga-3 ', R:5 '-gaattcccgg Ggatcctgatgttgacggcactgttga-3 '), using the genomic DNA of the big grain in parent river as template amplification 4073bp's OsGRF4 full length DNA (does not include promoter sequence), and amplified production is identified by agarose electrophoresis, and size is cut after meeting target Glue recycling.Then target fragment is connect with clone's carrier T (pEASY-Blunt Cloning Kit) of Quan Shi King Company: 1 μ L PCR recovery product adds the water of 1 μ L carrier T and 3 μ L, reacts at room temperature 5min.The competence that connection product is all added to 50 μ L is thin Born of the same parents, are gently mixed even, place 30min on ice.Then 42 DEG C of water-bath heat shock 30s, are placed on 2min on ice at once, and 250 μ are then added The LB culture medium of L, 200rpm is in 37 DEG C of culture 1h.Then LB culture medium is centrifuged 1min by 2000rpm, removes a part of supernatant Liquid retains 100 μ L or so, and gently suspension bacteria liquid, is all applied in the LB solid medium tablets added with antibiotic, trains at 37 DEG C It supports and is incubated overnight in case.After second day long good single bacterial plaque, picks them separately 20 bacterial plaques and be inoculated with into added with LB liquid medium Test tube in, cultivate 12h in 37 DEG C of incubators, etc. bacterium solutions muddiness when, be bacterium solution PCR respectively, whether detection target fragment correct It is connected into carrier T, M13 primer (M13F:5 '-GTAAAACGACGGCCAGT-3 ', M13R:5 '-in primer carrier T CAGGAAACAGCTATGAC-3′).Bacterium solution PCR is correctly cloned and is sequenced, analyze target gene sequence whether have it is prominent Become, the right-on clone of sequence connects for subsequent expression vector.With BamH I, single endonuclease digestion is connected with the T of target gene respectively Carrier and the pCUbi1390 expression vector (Fig. 2A) driven by Ubiquitin promoter are tried using Clontech Infusion Agent box merges target fragment with the carrier of linearisation, transformed competence colibacillus cell, chooses positive monoclonal plasmid and is sequenced.

Rice transformation and phenotypic evaluation: converting Agrobacterium competence for the correct plasmid of the sequence of above-mentioned building, Then pass through Agrobacterium-mediated transformation rice material NIL-Osgrf4.Agrobcterium-mediated transformation main process: Rice material by callus induction, callus subculture, pre-incubation, Agrobacterium culture, Agrobacterium is infected, callus is washed Culture is washed and selected, the differentiation of callus, takes root, practice seedling, transplanting, finally obtaining transgenic rice plant, (woods is supported the army, and is made Object journal, 2002,28:294-300).28 plants of overexpression transgenosis T0 are always obtained for rice plant, to T0 for transgenic seedling into Row hygromycin gene PCR detects (F:5 '-acggtgtcgtccatcacagtttgcc-3 '；R:5′- Ggaagtgcttgacattggggagttt-3 '), discovery transgenic positive single plant can expand target stripe, and transgene negative list Strain, water and non-transgenic negative control can not expand target stripe (Fig. 2 B).In addition, to the OsGRF4 gene of transgenic positive seedling Sequencing analysis is carried out, transgene negative single plant is TC in the base of target site as the result is shown, and transgenic positive single plant is AA It is bimodal (Fig. 2 C) with the heterozygous of TC.Sequencing result and hygromycin gene PCR testing result are completely the same, and 18 plants of final acquisition is super Express positive transgenic single plant.Transgenic paddy rice strain is overexpressed to OsGRF4 and NIL-Osgrf4 plant carries out cold treatment, knot The survival rate of OsGRF4 overexpression transgenic line is significantly higher than the survival rate of wild type NIL-Osgrf4 after fruit display cold treatment, The survival rate (Fig. 3) for increasing about 44% demonstrates again that gene OsGRF4 has the function of that enhancing plant is cold-resistant.Above-mentioned cold treatment side Method is referring to embodiment 1.

The building of 3 MicroRNA interference vector of embodiment and rice transformation

Size is obtained as the MicroRNA segment of 290bp using following primer amplification by template of pNW55 carrier:

I miR-s:5 '-agTAAAACGTTGACATCTCCCTTcaggagattcagtttga-3 '；

II miR-a:5 '-tgAAGGGAGATGTCAACGTTTTActgctgctgctacagcc-3 '；

III miR*s:5 '-ctAAGGGTGATCTCAACGTTTTAttcctgctgctaggctg-3 '；

IV miR*a:5 '-aaTAAAACGTTGAGATCACCCTTagagaggcaaaagtgaa-3 '.

The MicroRNA segment of 290bp is added into PstI and SpeI restriction enzyme site, double digestion target fragment and carrier PCUbi1390, is then attached reaction, and transformed competence colibacillus cell chooses positive monoclonal sequencing, the correct clone's matter that will be obtained Grain is imported into rice material NIL-OsGRF4 by the rice transformation system of mediated by agriculture bacillus.18 plants of T0 generation plants are obtained Strain carries out hygromycin gene PCR detection and sequencing analysis to transgenic seedling, finally obtains the positive single plant of 10 plants of interference.It is right OsGRF4 inhibits express transgenic rice strain and NIL-OsGRF4 plant to carry out cold treatment, as the result is shown OsGRF4 after cold treatment Inhibit the survival rate of express transgenic strain to be substantially less than the survival rate of wild type NIL-OsGRF4, reduces by about 44% survival rate (Fig. 4) is also demonstrated that gene OsGRF4 is adjustable the cold resistance of plant.The rice transformation of above-mentioned mediated by agriculture bacillus is referring to real The method for applying example 2, above-mentioned cold treatment method is referring to embodiment 1.

4 gene OsGRF4 of embodiment is cultivating the application in cold-resistant New Crop Varieties

During cultivating New Crop Varieties, transgenic method can be used, using including gene OsGRF4 of the present invention Plant expression vector transformation receptor crop and obtain genetically modified crops plant, and then the cold resistance of receptor crop can be improved. Crop plant containing gene OsGRF4 provided by the invention can also be hybridized with another crop plant and obtain progeny plant plant Strain, its cold resistance of progeny plant plant obtained are improved.The crop includes rice, corn, sorghum, wheat and barley Deng.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Hunan Research Centre for Hybrid Rice

The Institute of Subtropical Agriculture, The Chinese Academy of Sciences

<120>paddy gene OsGRF4 is improving the application in plant cold tolerance

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Met Thr Met Pro Tyr Ala Ser Leu Ser Pro Ala Val Ala Asp His Arg

1 5 10 15

Ser Ser Pro Ala Ala Ala Thr Ala Ser Leu Leu Pro Phe Cys Arg Ser

20 25 30

Thr Pro Leu Ser Ala Gly Gly Gly Val Val Ala Met Gly Glu Asp Ala

35 40 45

Pro Met Thr Ala Arg Trp Pro Pro Ala Ala Ala Ala Arg Leu Pro Pro

50 55 60

Phe Thr Ala Ala Gln Tyr Glu Glu Leu Glu Gln Gln Ala Leu Ile Tyr

65 70 75 80

Lys Tyr Leu Val Ala Gly Val Pro Val Pro Pro Asp Leu Val Leu Pro

85 90 95

Ile Arg Arg Gly Leu Asp Ser Leu Ala Ala Arg Phe Tyr Asn His Pro

100 105 110

Ala Leu Gly Tyr Gly Pro Tyr Phe Gly Lys Lys Leu Asp Pro Glu Pro

115 120 125

Gly Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg Cys Ser Lys Glu

130 135 140

Ala Ala Pro Asp Ser Lys Tyr Cys Glu Arg His Met His Arg Gly Arg

145 150 155 160

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

165 170 175

Pro Pro Ser Ser Val Val Gly Ser Ala Ala Ala Pro Leu Ala Ala Ala

180 185 190

Ser Asn Gly Ser Ser Phe Gln Asn His Ser Leu Tyr Pro Ala Ile Ala

195 200 205

Gly Ser Asn Gly Gly Gly Gly Gly Arg Asn Met Pro Ser Ser Phe Gly

210 215 220

Ser Ala Leu Gly Ser Gln Leu His Met Asp Asn Ala Ala Pro Tyr Ala

225 230 235 240

Ala Val Gly Gly Gly Thr Gly Lys Asp Leu Arg Tyr Thr Ala Tyr Gly

245 250 255

Thr Arg Ser Leu Ala Asp Glu Gln Ser Gln Leu Ile Thr Glu Ala Ile

260 265 270

Asn Thr Ser Ile Glu Asn Pro Trp Arg Leu Leu Pro Ser Gln Asn Ser

275 280 285

Pro Phe Pro Leu Ser Ser Tyr Ser Gln Leu Gly Ala Leu Ser Asp Leu

290 295 300

Gly Gln Asn Thr Pro Ser Ser Leu Ser Lys Val Gln Arg Gln Pro Leu

305 310 315 320

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

325 330 335

Asn Gln Thr Leu Arg Pro Phe Phe Asp Glu Trp Pro Lys Gly Arg Asp

340 345 350

Ser Trp Ser Asp Leu Ala Asp Glu Asn Ala Asn Leu Ser Ser Phe Ser

355 360 365

Gly Thr Gln Leu Ser Ile Ser Ile Pro Met Ala Ser Ser Asp Phe Ser

370 375 380

Ala Ala Ser Ser Arg Ser Thr Asn Gly Asp

385 390

<210> 4

<211> 2101

<212> DNA

<213>rice (Oryza sativa)

<400> 4

gttttctttt catcgtagtt tttcttgcag ccggttttca aaccactagt attatagata 60

taattttttt atttgcaatt tattttttac gatttatcaa ccgcagttta tccgattagt 120

ctttggaaaa tgttactgga agaagtaaaa cccagataac ccacaccaaa taatattaat 180

aaaaaatctg catctgcatt atagtacgtc aatccagctc cacatgatac tgatcttgtg 240

gtactgtagt agtaatagta ctccctccgt ccgtcctagt atagtggacg tttccattca 300

ttttatttga aaaattagta caaatataaa aaaagataag tcatatgtaa aatattttga 360

taataaaaca attgacaaac aaaataaata ataattctaa aattttttga ataaaacgaa 420

tgatcaaaaa ttataaacaa aaacttaaga agacaagtaa tatgggacgg aggtagttgt 480

agtatattag tacactgttt tgaggtattc caacgtcgaa taaaacagag aagtaccaca 540

ccattattgt gaactcgaaa cctggtagtg attaattgcc tcatggcaag caaactgaaa 600

caaactacta ttactactgc tctcccgttt tatattgttc atcgtataac ccaaaatcag 660

aatttccaaa ttatatcttg taatcttgac tgcatcgttt gacatacaat actattaaat 720

ctatacatat aaattgagtc tgtatacgta tatacaagca cttgagatgg ttaagcactt 780

tttttagcat tataagtttc ttattttgta ggatttttag tacgaggtaa gacatacttg 840

aaaaaaattc taagaactag agtgcatgtg accacctaac tccttgcaat ttttattctt 900

ataatttgaa aatcctataa accaaataag cccttcaaag gaaattaaat catgaggttt 960

gaggttaggt ttgaattctc taaaaagtgg aggaaaggac tcaacagaaa aaaaaaatcc 1020

tatagaattt cgatcctata aaattttagt taaaaatact ttgttccaaa attgccatgg 1080

ataaaatgta atttctatgc atacgactaa attatcgatg gcaacagtgc atgagcatat 1140

atttatttca ttgacctacg gttgcatgtc tccgatctct atggagtagt accgagacta 1200

agtttagttt caaacttttc cttcaaacat acagcttttt tatcacatta aaactttcct 1260

acgtataaac tttcaacttt tccatcacat ctttcaattt caaccaaact tttaatttta 1320

acgtgaacta aacacaccct gaattcaaaa ctctttttat tttccttcaa gatgtccgat 1380

gcacacgctc tatgtagacg caagaagatg ttggagcagc agactaacag tagcaaaaaa 1440

atggcaggtc gaaaagcaac tgcgacggtt gctccgtcat cctctcatcg cctttttatt 1500

gctccggcgt tgggaaccgc aacaatggaa cagcccaaat cgacagtccc ctcccccccc 1560

tcccccatcc tctctctccc cacgcaatac ttgtcactac tcgcgctgct cactacagcg 1620

tctctgcatg tatatccatc tatccatcca ttcccccatt ttccaaataa aaatacagca 1680

aaccaaacac aaacgcagcc tcgcactgta ctcgaagaaa aatcggtgct gtacgtacta 1740

cgccacgaga taacgagaga gagagagaga gagagagaga ggagaaaatg gaaatgctac 1800

tgctcgtacc acgccgctac gtccgctagg tcgacaggcc cggggggagg caggtgtttg 1860

tcgtctagct cgggtcggag cgcgccttct cgtgtcgggc tcgacgtccg cgactcctcg 1920

cccctggtcg agagctcgca ggcgcagcgg gagagagaga gagagagaga gagacaagcc 1980

gcgcaataaa ggcgcgcgcg cgagcgagcg aagcaaagca ccattactaa agaccgcggc 2040

gtgtgcttgc gttgcgagcg agcgagagcg agagagagat tgagagagag agagggaagg 2100

g 2101

Claims (8)

1. paddy gene OsGRF4 is improving the application in plant cold tolerance, which is characterized in that the paddy gene OsGRF4 is Encode following protein (a) or gene (b):

(a) protein that the amino acid sequence shown in SEQ ID NO:3 forms；

(b) sequence shown in SEQ ID NO:3 be substituted, lack or add one or several amino acid and with same function by (a) protein derived from.

2. application according to claim 1, which is characterized in that the plant includes rice, corn, sorghum, wheat and big Wheat.

3. a kind of method for improving plant cold tolerance, which is characterized in that the described method includes:

1) making plant includes paddy gene OsGRF4；Or

2) plant is made to express paddy gene OsGRF4；

Wherein, the definition of paddy gene OsGRF4 is the same as described in claim 1.

4. according to the method described in claim 3, it is characterized in that, the method includes transgenosis, hybridization, backcrossing, selfing or Vegetative propagation.

5. according to the method described in claim 3, it is characterized in that, the method includes using Ti-plasmids, plant viral vector, Directly delivered DNA, microinjection, particle gun, conductance, the method for mediated by agriculture bacillus are by the recombination table comprising paddy gene OsGRF4 Up to vector introduction plant cell, transgenic plant is obtained.

6. according to the method described in claim 3, it is characterized in that, which comprises will contain paddy gene OsGRF4's Plant hybridizes with another plant, obtains filial generation.

7. according to the described in any item methods of claim 3-6, which is characterized in that the plant include rice, corn, sorghum, Wheat and barley.

8. the promoter of paddy gene OsGRF4, which is characterized in that the promoter are as follows:

I) nucleotide sequence shown in SEQ ID NO:4；

Ii) nucleotide sequence shown in SEQ ID NO:4 is substituted, lacks and/or increases one or more nucleotide and has The nucleotide sequence of promoter activity；

Iii) hybridize and have under strict conditions the nucleotide sequence of promoter activity, institute with sequence shown in SEQ ID NO:4 Stating stringent condition is to hybridize at 65 DEG C, in 6 × SSC solution containing 0.5%SDS then with 2 × SSC containing 0.1%SDS Solution, it is primary that 1 × SSC solution containing 0.1%SDS respectively washes film；Or

Iv) and i), ii) or nucleotide sequence iii) have 70% or more, preferably 80% or more, more preferable 90% or more is homologous Property and with promoter activity nucleotide sequence.