CN101289502A - Plant frigostabile protein, encoding gene thereof and applications - Google Patents

Abstract

The invention discloses a plant low temperature resistant protein and encoding gene and application thereof. The protein has one of the following amino acid residue sequences: 1) an amino acid residue sequence of SEQ ID No.3 in a sequence table; 2) a protein with plant low temperature resistant protein activity, which is gained by substituting and/or deleting and/or adding one or a plurality of amino acid residues in the SEQ ID No.3 amino acid residue sequence in the sequence table. The encoding gene of the low temperature resistant protein of the invention can be transferred to a plant to enhance frost resistance thereof. The gene not only has important theoretic and practical significance for the research on the cold (frost) resistance molecular mechanism of plants and the breeding of stress resistant plants, but also provides an economical, fast and effective way to improve the cold resistance of plants. The plant low temperature resistant protein has wide application space and market prospect in the agriculture field.

Description

A kind of plant frigostabile protein and encoding gene thereof and application

Technical field

The present invention relates to a kind of plant frigostabile protein and encoding gene thereof and application.

Background technology

Low temperature stress is an abiotic stress common in the Rice Production.According to statistics, the whole world has the rice more than 1,500 ten thousand hectares to be subjected to the low temperature threat, and only China will lose hundred million kilograms of the about 50-100 of paddy every year on average in famine year, bring serious harm for people's productive life.When plant meets with low temperature stress and scarce phosphorus, all can influence the growth and the division of cell, the branch of tillering is reduced, poor growth, plant is short and small even dead, has a strong impact on plant-growth and growth.Therefore in paddy rice, excavate simultaneously and coerce relevant excellent gene with low temperature resistant and responding low-phosphor, and they are applied to have important theoretical meaning and more practical value in the rice breeding production, also be an effective way that solves a current rice breeding difficult problem.

Have some and studies show that the gene that contains SPX (SYG1/Pho81/XPR1) structural domain participates in the signal transduction and the adjusting approach of phosphorus.SPX is about 180 amino acid longs, is present in a structural domain of 3 kinds of known proteins (SYG1, Pho81 and XPR1) N-terminal, and its title derives from this three kinds of proteic initials.As far back as nineteen ninety-five, people such as Spain (Spain, B.H., Koo, D., Ramakrishnan, M., Dzudzor, B.and Colicelli, J. (1995) Truncatedforms of a novel yeast protein suppress the lethality of a G protein alpha subunit deficiencyby interacting with the beta subunit.J Biol Chem, 270, thus discover that 25435-25444) N-terminal of yeast SYG1 can suppress the signal transduction of mating pheromone (mating pheromone) with the G-protein binding; Under the phosphate starvation condition, there is report CDK to suppress enzymic activity (Lee, the M. that sub-Pho81 can suppress PHO80-PHO85 and Pcl7-Pho85 complex body, O ' Regan, S., Moreau, J.L., Johnson, A.L., Johnston, L.H.and Goding, C.R. (2000) Regulation of the Pcl7-Pho85 cyclin-cdk complex by Pho81.Mol Microbiol, 38,411-422); People such as Poleg (1996) (Poleg, Y., Aramayo, R., Kang, S., Hall, J.G.andMetzenberg, R.L. (1996) NUC-2, a component of the phosphate-regulated signaltransduction pathway in Neurospora crassa, is an ankyrin repeat protein.Mol Gen Genet, 252,709-716) in fungi Neurospora crassa, find, but with the existence of the NUC-2 albumen perception phosphorus of PHO81 structural similitude and participate in the adjusting approach of phosphorus transporter.2002, people such as Hamburger (Hamburger, D., Rezzonico, E., MacDonald-Comber Petetot, J., Somerville, C.and Poirier, Y. (2002) Identification and characterization of the Arabidopsis PHO1 gene involved in phosphateloading to the xylem.Plant Cell, 14,889-902) in Arabidopis thaliana, also find a class SPX gene, i.e. PHO1 (having SPX structural domain and EXS structural domain) gene and PHO1 albuminoid, they can participate in phosphorus by the transportation of root to stem, and in the microtubule fasolculus cell of root specifically expressing.

Summary of the invention

The purpose of this invention is to provide a kind of plant frigostabile protein and encoding gene thereof.

Plant frigostabile protein provided by the present invention, name is called OsSPX3, derives from Oryza paddy rice (Oryza sativaL.), is following (a) or protein (b):

(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 3;

(b) with the amino acid residue sequence of sequence in the sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the plant frigostabile function by (a) deutero-protein.

Wherein, the protein sequence shown in the sequence 3 in the sequence table is made up of 277 amino-acid residues, is the SPX protein structure domain from aminoterminal (N end) 1-159 amino acids residue.

In order to make OsSPX3 in (a) be secreted in cell pericentral siphon or the substratum or to make its function-stable, proteinic N end that can the amino acid residue sequence of sequence 2 is formed in by sequence table connects signal peptide sequence, for the OsSPX3 in (a) is convenient to purifying, proteinic N end or C end that can the amino acid residue sequence of sequence 3 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	11	EQKLISEEDL

Above-mentioned (b) but in the OsSPX3 synthetic, also can synthesize its encoding gene earlier, carry out biology according to following method again and express and to obtain.The encoding gene of OsSPX3 in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna of sequence in the sequence table 1 or sequence 2, and/or carry out the missense mutation of one or several base pair, and/or at the encoding sequence of its 5 ' end attach signal peptide, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The encoding gene of above-mentioned plant frigostabile protein (OsSPX3) also belongs to protection scope of the present invention.

The cDNA gene of above-mentioned plant frigostabile protein can have one of following nucleotide sequence:

1) dna sequence dna of sequence 2 in the sequence table;

2) polynucleotide of protein sequence shown in the sequence 3 in the code sequence tabulation;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with sequence in the sequence table 2.

Wherein, sequence 2 is made up of 834 deoxynucleotides in the sequence table, and 5 of sequence 2 ' end 1-834 position nucleotides sequence is classified encoding sequence (ORF) as in sequence table, and coding has the protein of the amino acid residue sequence of sequence 3 in the sequence table.

The genomic gene of above-mentioned plant frigostabile protein can have one of following nucleotide sequence:

1) dna sequence dna of sequence 1 in the sequence table;

2) polynucleotide of protein sequence shown in the sequence 3 in the code sequence tabulation;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with sequence in the sequence table 2.

Wherein, sequence 1 is made up of 1993 deoxynucleotides in the sequence table, 5 of sequence 1 ' end 1-237 position nucleotides sequence is classified first exon as in sequence table, 5 of sequence 1 ' end 328-478 position nucleotides sequence is classified second exon as in sequence table, and 5 of sequence 1 ' end 1548-1993 position nucleotides sequence is classified the 3rd exon as in sequence table; 5 of sequence 1 ' end 238-327 position nucleotides sequence is classified first intron as in sequence table, and 5 of sequence 1 ' end 479-1547 position nucleotides sequence is classified second intron as in sequence table.

The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

The expression vector, transgenic cell line and the host bacterium that contain the encoding gene of above-mentioned plant frigostabile protein all belong to protection scope of the present invention.

The present invention also provides the application of above-mentioned plant frigostabile protein encoding gene in improving the plant frost resistance.

The method of described raising plant frost resistance is that above-mentioned plant frigostabile protein encoding gene is changed in the plant by plant expression vector, and screening obtains the plant that frost resistance improves.

Above-mentioned plant expression vector can be pCAMBIA1300, pBI121, pBin19, pCAMBIA2301 or pCAMBIA1301-UbiN or other plant expression vector of deriving.Described plant host both can be monocotyledonss such as paddy rice, corn, wheat, also can be dicotyledonss such as tomato, Arabidopis thaliana, tobacco, cotton; Be preferably paddy rice, tobacco.

When using OsSPX3 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) etc., they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Carry the present invention encode the gene OsSPX3 that improves the tobacco frost resistance plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method rice transformation cell or tissue such as agriculture bacillus mediated by using, and rice cell or the tissue cultivating that transforms become plant.

The encoding gene of frigostabile protein of the present invention can change over to and improve the plant frost resistance in the plant.This gene is for plant cold-resistant (freezing) Molecular Study, and the seed selection of stress resistance of plant kind has important theory and practical significance, and provides an economy, approach fast and effectively for the resistance to cold of improvement crop.The present invention has wide application space and market outlook at agriculture field.

Description of drawings

Fig. 1 be rice Os SPX3 gene under the subzero treatment condition with normal condition under (contrast) expression amount relatively.

Fig. 2 is that the design of graphics (A among the figure) of plant expression vector (pCOU OsSPX3) of rice Os SPX3 and the RT-PCR that changes pCOU OsSPX3 tobacco identify.

Fig. 3 is for changeing the phenotype analytical of pCOU OsSPX3 tobacco under differing temps.

Fig. 4 is for changeing the variation of the physical signs (comprise iontophoretic injection, proline content, sucrose content and phosphorus content) of pCOU OsSPX3 tobacco under differing temps.

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and the primer synthesizes and examining order is finished by Beijing AudioCodes biotechnology limited liability company.

Embodiment 1, the paddy rice SPX gene expression characteristic under the subzero treatment condition detects

Excavate by chip data, in the rice genome sequence, find different another kind of of the PHO1 subfamily of a prediction to have the SPX structural domain and do not have the SPX gene of EXS structural domain, be positioned No. 10 karyomit(e) from 12,757,421bp to 12,760,157bp, its NCBI location number is NP_001064515, and NCBI gene number (GENEID) is: Os10g0392600, TIGR number is LOC_Os10g25310.

Utilize the method for real-time quantitative RT-PCR that the express spectra of gene under the deepfreeze condition of above-mentioned prediction verified.According to the information design real-time quantitative RT-PCR primer of the genome database of NCBI, forward primer Os-s10F:5 '-tcgccggacatggaaaggat-3 '; Reverse primer Os-s10R:5 '-cggcggcagcgagaacc-3 '.

Fine with Japan is material, carry out real-time quantitative RT-PCR according to following method: will germinate about 1 week, the Japanese fine seedling that bud is about 5mm places 4-5 ℃ to carry out subzero treatment, treatment time is 6h, 12h, 24h is behind the 48h, utilizing TRIZOL reagent to extract RNA, is contrast with the seedling under the normal growth condition.With this RNA is template, use the M-MLV ThermoScript II to carry out reverse transcription and obtain cDNA, and be template with this cDNA, be primer with Os-s6F and Os-s6R, carry out real-time quantitative RT-PCR and detect.Real-time quantitative RT-PCR detection internal standard gene is selected the 18s rRNA gene in the paddy rice for use, and primer sequence is: 18S-F:5 '-GCTTTGGTGACTCTAGATAAC-3 '; 18S-R:5 '-GTCGGGAGTGGGTAATTTGC-3 '.

Reaction system is: 4 μ l cDNA templates (2ng/ μ l), 4.5 μ l SYBR Green Master Mix, 1 μ l special primer, 0.5 μ l ddH ₂O.

Amplification program following (detection system of utilizing MJ Research company to produce): 95 ℃ of 5min, 1 circulation, 95 ℃ of 30s, 60 ℃ of 30s, read plate (read plate), 85 ℃ of 1s read plate (read plate), totally 40 circulations, 72 ℃ of 2min make solubility curve every 0.2 ℃ for 65 ℃-95 ℃.3 repetitions are established in above-mentioned experiment.

The result as shown in Figure 1, the result shows, above-mentioned paddy rice SPX gene is at subzero treatment 6h, 12h, behind 24h and the 48h, expression amount continues to increase, and illustrates that this gene is cold induced gene really.The ordinate zou of Fig. 1 is represented be subzero treatment paddy rice SPX gene expression amount with normal condition under the ratio of paddy rice SPX gene expression amount.

The acquisition and the functional verification thereof of embodiment 2, paddy rice SPX gene and proteins encoded thereof

One, the acquisition of paddy rice SPX gene and proteins encoded thereof

The full length cDNA sequence of design implementation example 1 described paddy rice SPX gene designs primer, and introduces restriction enzyme BglII and KpnI recognition site and protection base respectively at the primer two ends, and primer sequence is as follows: CDS-PF:5 '- CGGGGTACCCCGATGAAGTTTGGGAAGAGGCTGAAG-3 ' (band underscore base is restriction enzyme KpnI recognition site and protection base); CDS-PR:5 '- CGAGATCTCGTTAGGCATAAAAAAACTGTAAACTTGGAAT-3 ' (band underscore base is restriction enzyme BglII recognition site and protection base).Utilize TRIZOL reagent extract Japanese fine subzero treatment 12h (4 ℃) the bud phase plant RNA, with this RNA is template, use the M-MLV ThermoScript II to carry out reverse transcription and obtain cDNA, with this cDNA is template, under the guiding of primer CDS-PF and primer CDS-PR, cDNA sequence with conventional PCR method amplifying rice SPX gene, after reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, recovery and purifying obtain the dna fragmentation of 834bp, and this fragment is checked order, and the result shows, the fragment that pcr amplification obtains has the nucleotide sequence of sequence 2 in the sequence table, with its called after OsSPX3.The nucleotide sequence of sequence 2 is made up of 834 deoxynucleotides in the sequence table, and 5 of sequence 2 ' end 1-834 position nucleotides sequence is classified encoding sequence (ORF) as in sequence table, and coding has the protein of the amino acid residue sequence of sequence 3 in the sequence table.With this albumen called after OsSPX3.The protein sequence shown in the sequence 3 has 277 amino acid in the sequence table, is the SPX protein structure domain from aminoterminal (N end) 1-159 amino acids residue.Pass through sequence alignment, compare with TIGR rice genome database, confirm that its location is number for LOC_Os10g25310, consistent with the gene of being predicted among the embodiment 1, its genomic gene has the nucleotide sequence of sequence 1 in the sequence table, 5 of sequence 1 ' end 1-237 position nucleotides sequence is classified first exon as in sequence table, 5 of sequence 1 ' end 328-478 position nucleotides sequence is classified second exon as in sequence table, and 5 of sequence 1 ' end 1548-1993 position nucleotides sequence is classified the 3rd exon as in sequence table; 5 of sequence 1 ' end 238-327 position nucleotides sequence is classified first intron as in sequence table, and 5 of sequence 1 ' end 479-1547 position nucleotides sequence is classified second intron as in sequence table.

Two, the low temperature resistant functional verification of paddy rice SPX gene and proteins encoded thereof

1, the structure of the plant expression vector of rice Os SPX3 (pCOU OsSPX3)

The fragment that the step 1 pcr amplification is obtained has the cDNA fragment of the OsSPX3 of the nucleotide sequence of sequence 2 in the sequence table, carry out double digestion with BglII and KpnI, after reclaiming purifying, be inserted into plant expression vector pCambial301-UbiN (GenBank number: AF234296) between the BglII of multiple clone site and the KpnI restriction enzyme site, obtain recombinant expression vector, this recombinant expression vector is carried out the enzyme evaluation of cutting and check order, evaluation is shown the recombinant expression vector called after pCOU OsSPX3 (its structure diagram is shown in A among Fig. 2) of the nucleotide sequence that contains sequence 2 in the ordered list.

2, change the acquisition of OsSPX3 tobacco

1) transformation of tobacco

The plant expression vector pCOU OsSPX3 of the rice Os SPX3 that step 1 is made up is with the explant of Agrobacterium-mediated Transformation method transformation of tobacco (Nicotiana tabacum L.cv.Xanthinn).Concrete grammar is:

(1) pCOU OsSPX3 is transformed the Agrobacterium that Agrobacterium obtains containing pCOU OsSPX3, the Agrobacterium that will contain pCOU OsSPX3 is inoculated in the YEB liquid medium (100 μ g/ml Kan, 75 μ g/ml Rif), and 28 ℃ of shaking culture are to OD ₆₀₀Be 0.6-0.8; With 10, the centrifugal 1min of 000rpm room temperature, with MS salts solution (pH7.0) thalline that suspends again, the 20-50 that adopts the MS salts solution to be diluted to original volume during use doubly obtains containing the Agrobacterium bacterium liquid of pCOUOsSPX3.

(2) aseptic tobacco (Nicotiana tabacum L.cv.Xanthinn) blade cuts edge and main vein, is cut into 0.4 * 0.6cm ²Size obtains explant;

(3) explant that step (2) is obtained soaks 10min in the Agrobacterium bacterium liquid that contains pCOU OsSPX3 that step (1) obtains;

(4) blot the bacterium liquid on explant material surface with aseptic filter paper, change the MS minimum medium of upper berth one deck filter paper over to, 28 ℃ of dark cultivations; After 3 days, material forwarded to contain in the corresponding antibiotic MS division culture medium cultivate; Treat that resistant buds grows to 2-3cm when high, downcut budlet and change root induction in the MS root media over to that differentiation obtaining transfer-gen plant.

2) PCR of transgene tobacco identifies

Extraction step 1) genomic dna of the transgene tobacco that obtains and as template, at primer 1:5 '-ATGAAGTTTGGGAAGAGGCTGAAG-3 ' and primer 2: 5 '-guiding of TTAGGCATAAAAAAACTGTAAACTTGGAAT-3 ' under, carry out PCR and detect, the PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30s then, 58 ℃ of 45s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ of 10min.After reaction finishes, amplified production is carried out 1% agarose gel electrophoresis detect, detected result is shown in B among Fig. 2, and positive OsSPX3 transfer-gen plant can amplify the band of about 834bp.The result obtains 6 strain PCR and identifies that male changes pCOU OsSPX3 tobacco T0 for plant.The swimming lane WT of B is the wild-type tobacco contrast among Fig. 2, and other swimming lane is for changeing pCOU OsSPX3 tobacco PCR qualification result, and the actin among Fig. 2 among the B represents the Actin muscle in the microfilament, here the contrast that detects as transgenosis.

3, change the low temperature resistant functional verification of pCOU OsSPX3 tobacco

Step 2 with step 2) PCT identifies that male changes pCOU OsSPX3 tobacco and wild-type tobacco (WT, Nicotiana tabacum L.cv.Xanthinn) normal (25 ℃, 16 hours daytime/8 hour cycles at night) 4 weeks of growth, place 0 ℃ then respectively, cultivated 24 hours, and then-2 ℃ of freezing treatment 3 hours, and then place normal (25 ℃) condition to recover to grow 2 days, in this process, observe the upgrowth situation that changes pCOU OsSPX3 tobacco and wild-type tobacco, detected their iontophoretic injection, proline content, sucrose content and blade phosphorus content simultaneously.

The upgrowth situation result as shown in Figure 3, the result shows, in the time in normal 4 weeks of cultivation, change the growing state no significant difference (A among Fig. 3) of pCOU OsSPX3 tobacco and wild-type tobacco, when they being placed 0 ℃ after 24 hours, the wild-type plant changes pCOU OsSPX3 tobacco plant obviously wilt (B among Fig. 3); When with them again-2 ℃ of freezing treatment after 3 hours, though wild-type and transfer-gen plant all show freezing injury, the wild-type tobacco plant is more obvious, all plant almost completely wilt (C among Fig. 3); Recovery under normal condition two days later growth again, the part transfer-gen plant shows the growth recovery sign, and most wild-type plant can't recover (D among Fig. 3).WT among Fig. 3 among A, B, C, the D all represents above-mentioned wild-type tobacco, and Ubi::OsSPX3 all represents to change pCOU OsSPX3 tobacco.

The physiological and biochemical relevant (iontophoretic injection, proline content, sucrose content, phosphorus content) detected result with cold resistance as shown in Figure 4, the result shows that changeing the OsSPX3 genetic tobacco has stronger frost resistance (Fig. 4) than wild-type tobacco.Simultaneously, the mensuration of blade phosphorus content shows that under cold condition the decline degree of phosphorus content is greater than wild-type tobacco (Fig. 4) in the plant of transgene tobacco, and A is for changeing the specific conductivity detected result of pCOU OsSPX3 tobacco among Fig. 4; B is for changeing the proline content detected result of pCOU OsSPX3 tobacco among Fig. 4; C is for changeing the sucrose content detected result of pCOU OsSPX3 tobacco among Fig. 4; D is for changeing the phosphorus content detected result of pCOU OsSPX3 tobacco among Fig. 4.WT among Fig. 4 among A, B, C, the D all represents above-mentioned wild-type tobacco, and Ubi::OsSPX3 all represents to change pCOU OsSPX3 tobacco.FW among Fig. 4 represents fresh weight, and DW represents dry weight.

Sequence table

<160>3

<210>1

<211>1993

<212>DNA

＜213〉Oryza paddy rice (Oryza sativa L.)

<400>1

atgaagtttg ggaagaggct gaagaagcag gtggaggaga gcctcccgga gtggagggac 60

aagttcttgg cgtacaagcg cctcaagaag ctcgtcaggc tcgtctcctc ctcctccggc 120

gatgtcggcg gcggcggcgg cggcgaggcc gaattcgtgc ggctgctcga cggcgaggtc 180

gacaggatca acgccttctt cctcgagcag gaggaggagt tcgtcatacg gcagagggtc 240

tctgcattag ctatttctgc agcaaagatt tttggttttg ggaagttgag atgatgattg 300

tgagcctgcg tgcattgttc gtcgcaggag ctgcaggaga cagtggagaa ggtggccggc 360

ggcggcggtg gagggcggcg gccggcggcg gcggagatga ggagggtgag gaaggagatc 420

gtggacctgc acggggagat ggtgctgctg cttaactaca gcgccgtcaa ctacacaggt 480

ttgagtttgg tttcaaattt caaattcaaa cttaggaggt gaaaactttc agaattttcg 540

tatacgaccc gtgacaaggt tcgtacggag taattacgaa ctggtattcc caaagagatc 600

tcaattccca aatttcaaat atttcgatgg gcccgggtag agcaccatac cagggggaat 660

agttttttct ttttaaaatc tttttttttt ggcgcaaaca ggtgagcatc atttgtagta 720

gctgtgtgct taacaacttc tgatacaaca gtactcctcc gtcctaaaat aaatgcagtt 780

ttgcactatt cacattcaac gtttgaccgt tcgtcttatt taaacctttt ttatgattag 840

tatttttatt actattagat gataaaacat aaatagtact ttacgtgtga ctaaatattt 900

tcaaattttt tacaaatttt ttaaataaga cggacggtca aacattgggc acggatatcc 960

acggctacac ttattttagg acggatgtag cactaacagt gattttttga cttgaaaatt 1020

ttcttatgat ttgatcactg ttcaacttga gacttgtcgt agccgtcccc atgtgagtca 1080

acagtatctt caggctgcac attacagcca aaattttctc tacacaacaa aattcattca 1140

gtgcatttgc acagaacagt aactgcaacc taaagacaat ctgtttatcc atatagatta 1200

tagttagaca cgatgatgat ctgtttatcc acaaatatta tagactaatc gtcttcccta 1260

tcgatcggta agtgactagg ctgtgttcct tactgggttg gaacccactt tctccgcgcg 1320

aaaaacggaa cagtttatta gcacattatt aattaagtat tagctttttt aaaaaataaa 1380

ttaatttgat ttttttaaaa caactttcgt ataaaaactt ctaaaaaaat cacaccgttt 1440

agcagcgccg ataacaaagg ataagggttg ggaccatgct gaaacgaacg cagccagaga 1500

ggctgaacat ggtgatgaat tattgatgat gatctgtgaa attgcagggc tggccaagat 1560

cctgaagaaa tacgacaagc gcaccggccg cctcctccgg ctgccgttca tcgagaaggt 1620

gctccggcag ccgttcttca cgacggagct catctcgagg ctcgtccgcg actgcgaggc 1680

caccatggag gccatcttca cctcctccgt ggcgacgacg gcgatggccg gcgatcgccg 1740

gacatggaaa ggatgctccg gcgacgccgg gatggctccg atggcagacc agcagggcat 1800

cttccggaac accgtcgccg cgctggcgac gatgaaggag ctccggagcg ggagctcgac 1860

gtacgggcgg ttctcgctgc cgccgatggc ggcgccggcc tcgccggagt ccgacgtgct 1920

gcagtccatc cgatccgatc cccatctgaa agagaatgga agaattccaa gtttacagtt 1980

tttttatgcc taa 1993

<210>2

<211>834

<212>DNA

＜213〉Oryza paddy rice (Oryza sativa L.)

<400>2

atgaagtttg ggaagaggct gaagaagcag gtggaggaga gcctcccgga gtggagggac 60

aagttcttgg cgtacaagcg cctcaagaag ctcgtcaggc tcgtctcctc ctcctccggc 120

gatgtcggcg gcggcggcgg cggcgaggcc gaattcgtgc ggctgctcga cggcgaggtc 180

gacaggatca acgccttctt cctcgagcag gaggaggagt tcgtcatacg gcagagggag 240

ctgcaggaga cagtggagaa ggtggccggc ggcggcggtg gagggcggcg gccggcggcg 300

gcggagatga ggagggtgag gaaggagatc gtggacctgc acggggagat ggtgctgctg 360

cttaactaca gcgccgtcaa ctacacaggg ctggccaaga tcctgaagaa atacgacaag 420

cgcaccggcc gcctcctccg gctgccgttc atcgagaagg tgctccggca gccgttcttc 480

acgacggagc tcatctcgag gctcgtccgc gactgcgagg ccaccatgga ggccatcttc 540

acctcctccg tggcgacgac ggcgatggcc ggcgatcgcc ggacatggaa aggatgctcc 600

ggcgacgccg ggatggctcc gatggcagac cagcagggca tcttccggaa caccgtcgcc 660

gcgctggcga cgatgaagga gctccggagc gggagctcga cgtacgggcg gttctcgctg 720

ccgccgatgg cggcgccggc ctcgccggag tccgacgtgc tgcagtccat ccgatccgat 780

ccccatctga aagagaatgg aagaattcca agtttacagt ttttttatgc ctaa 834

<210>3

<211>277

<212>PRT

＜213〉Oryza paddy rice (Oryza sativa L.)

<400>3

Met Lys Phe Gly Lys Arg Leu Lys Lys Gln Val Glu Glu Ser Leu Pro

1 5 10 15

Glu Trp Arg Asp Lys Phe Leu Ala Tyr Lys Arg Leu Lys Lys Leu Val

20 25 30

Arg Leu Val Ser Ser Ser Ser Gly Asp Val Gly Gly Gly Gly Gly Gly

35 40 45

Glu Ala Glu Phe Val Arg Leu Leu Asp Gly Glu Val Asp Arg Ile Asn

50 55 60

Ala Phe Phe Leu Glu Gln Glu Glu Glu Phe Val Ile Arg Gln Arg Glu

65 70 75 80

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

85 90 95

Arg Pro Ala Ala Ala Glu Met Arg Arg Val Arg Lys Glu Ile Val Asp

100 105 110

Leu His Gly Glu Met Val Leu Leu Leu Asn Tyr Ser Ala Val Asn Tyr

115 120 125

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

130 135 140

Leu Leu Arg Leu Pro Phe Ile Glu Lys Val Leu Arg Gln Pro Phe Phe

145 150 155 160

Thr Thr Glu Leu Ile Ser Arg Leu Val Arg Asp Cys Glu Ala Thr Met

165 170 175

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

180 185 190

Arg Arg Thr Trp Lys Gly Cys Ser Gly Asp Ala Gly Met Ala Pro Met

195 200 205

Ala Asp Gln Gln Gly Ile Phe Arg Asn Thr Val Ala Ala Leu Ala Thr

210 215 220

Met Lys Glu Leu Arg Ser Gly Ser Ser Thr Tyr Gly Arg Phe Ser Leu

225 230 235 240

Pro Pro Met Ala Ala Pro Ala Ser Pro Glu Ser Asp Val Leu Gln Ser

245 250 255

Ile Arg Ser Asp Pro His Leu Lys Glu Asn Gly Arg Ile Pro Ser Leu

260 265 270

Gln Phe Phe Tyr Ala

275

Claims (10)

1, a kind of plant frigostabile protein is the protein with one of following amino acid residue sequences:

1) amino acid residue sequence of the SEQ ID № .3 in the sequence table;

2) with the SEQ ID № .3 amino acid residue sequence in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the active protein of plant frigostabile protein.

2, the encoding gene of the described plant frigostabile protein of claim 1.

3, encoding gene according to claim 2 is characterized in that: the cDNA gene of described plant frigostabile protein has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 nucleotide sequence;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

4, encoding gene according to claim 3 is characterized in that: the genomic gene of described plant frigostabile protein has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 nucleotide sequence;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

5, the recombinant expression vector that contains any described plant frigostabile protein encoding gene among the claim 2-4.

6, the transgenic cell line that contains any described plant frigostabile protein encoding gene among the claim 2-4.

7, the host bacterium that contains any described thing frigostabile protein encoding gene among the claim 2-4.

8, the application of any described plant frigostabile protein encoding gene in improving the plant frost resistance among the claim 2-4.

9, application according to claim 8, it is characterized in that: the method for described raising plant frost resistance is that any described plant frigostabile protein encoding gene changes in the plant by plant expression vector among the claim 2-4, and screening obtains the plant that frost resistance improves.

10, application according to claim 9 is characterized in that: described plant expression vector is pCAMBIA1300, pBI121, pBin19, pCAMBIA2301 or pCAMBIA1301-UbiN; Described plant is farm crop, is preferably paddy rice, tobacco.

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