CN101445553B - Paddy drought resistance gene OsSINAT5 and encoding protein and application thereof - Google Patents

Abstract

The invention provides a paddy drought resistance gene OsSINAT5 and an encoding protein and the application thereof. The encoding protein is the following protein (a) or (b): (a) a protein with the amino acid sequence shown in the sequence tables, such as SEQ ID NO. 2; or (b) a plant stress resistance-related protein derived from (a) by the substitution and/or deletion and/or addition of one or more amino acid residues to the amino acid sequence of (a). The plant stress resistance-related protein and the encoding gene thereof are applied to the culture of stress-resistant plants, particularly drought-resistant paddy.

Description

Paddy drought resistance gene OsSINAT 5 and proteins encoded thereof and application

Technical field:

The present invention relates to plant biotechnology field, be specifically related to separating clone, functional verification and the application of a paddy rice zinc finger protein gene dna fragmentation (gene).

Background technology:

Plant is because the biological characteristics of its set growth, make plant closely related with environment in growth and development process, and in process of growth, be subjected to the regulation and control of ambient signal inevitably and suffer from various environment stresses, as saline and alkaline, barren, thirsty fields, cold, freezing weather, the infringement of various pathogenic micro-organisms etc., and abiotic stress such as arid, saline and alkaline, high temperature are the major causes that causes crop production reduction in the world wide at present, and countries in the world government and scientific research personnel urgently wish to help to solve this significant problem by the biotechnology approach.Plant not only passes through physiological and biochemical procedure to the response and the adaptation of adverse circumstance, also need regulation and control by the molecular cell level, so the grow characteristic of research plant under adverse environmental factor, especially disclosing this specific character at molecular level, is that cultivation can be in the important foundation of the plant of normal growth growth under the adverse environmental factor; Utilize modern biotechnology improvement plant to obtain the prerequisite of the plant of strong stress resistance especially.

Environment-stress such as arid, high salt and low temperature have limited the raising of whole world grain yield greatly, and arid is the main factor of restriction agriculture production.According to statistics, arid occupy the first to the influence of world food crop in various natural disasteies, and its harm is equivalent to other natural disaster sum.Therefore, the understanding plant is improved the drought resistance of plant to the reaction mechanism of arid, is the important foundation of agricultural produce.Under adverse circumstances such as arid, plant can be made corresponding adjustment on molecule, cell and integral level, with the injury that farthest reduces environment and caused and survived.Arid mainly is growth, the damage vegetable cell that causes osmotic stress and suppress plant to the influence of plant, finally causes the degraded of DNA and causes the death of vegetable cell.Arid is to the influence of plant, and only some may be a kind of adaptive response of plant, and many reactions are the physiological damage results that cause because of coercing.The reacting phase of the osmotic stress that this process and plant cause high salt and low temperature seemingly.

Existing studies show that, osmotic stress can be induced or the expression of suppressor gene, the product of these genes not only can be participated in the stress response of plant directly, and can regulate other Expression of Related Genes or participate in signal transduction path, thereby plant is avoided or reduce injury, strengthen coercing the resistance of environment.Ubiquitin (Ubiquitin) be a kind of in eukaryote the ubiquitous small protein of forming by 76 amino acid, its aminoacid sequence and structure be high conservative all.The protein degradation of ubiquitin mediation is realized by ubiquitin/26S proteasome pathway.Its primary process is: the ubiquitin molecule is activated by ubiquitin activating enzyme (E1) under the situation that ATP exists, the C-terminal glycine of ubiquitin is attached on the cysteine sulfydryl of E1, transfer to then on the cysteine sulfydryl of ubiquitin binding enzyme (E2), at last under the effect of ubiquitin ligase (E3), covalently bound to the lysine residue of protein target molecule, form poly Ub-target protein mixture; Little peptide or amino acid are discerned and be degraded to this mixture by the 26S proteasome, discharges Ub simultaneously for recycle.Studies show that in a large number, is that organism is kept the important physiological and biochemical procedure that self normal growth is grown and conformed by the protein degradation of ubiquitin mediation.At present, in Arabidopis thaliana (Arabidopsis thaliana), identified XERICO (KO et al., 2006), SIZ1 (Catala et al., 2006), SDIR1 (Zhang et al., 2007), DRIP1 and DRIP2 (Qin et al., 2008) and PUB22 and PUB23 (Cho et al., 2008) etc. ubiquitin ligase is participated in the control of plant to arid reaction committed step directly, overexpression or suppress these expression of gene and can significantly strengthen the drought resistance of plant, these protein degradations that studies show that the ubiquitin mediation play an important role in the plant drought reaction.

Paddy rice is world's staple food crop, and the population in the whole world about 2/3rds is food with rice.And China is maximum rice producing country and country of consumption, and its rice does area and rice and makes ultimate production and account for the whole world 23% and 37% respectively, and it is very big that worldwide decrease in precipitation and skewness are produced influence to it.Moreover the annual water total amount of China is 5,000 hundred million m ³, agricultural water accounts for 70%, and paddy rice is first rich and influential family of agriculture water consumption, and about 54% of its quantity of water consumption whole nation total water amount accounts for more than 65% of agriculture total water amount.Continuous increase along with China's population, the fast development and the Ecological environment worsening of industry, China is being faced with more and more serious water shortage problem, seriously restricting the raising of China's grain yield dried morning, therefore carry out the anti-Journal of Sex Research early of paddy rice, significant for improving the anti-property early of crop and reducing agricultural production water.But the drought resisting transgenic paddy rice of cultivating in paddy rice at present is also less, therefore finds out ubiquitin ligase relevant with drought resisting in the paddy rice, and the kind of cultivating drought resisting is significant to improving rice yield.

Summary of the invention:

Technical problem to be solved by this invention is: at above-mentioned the deficiencies in the prior art, provide a kind of paddy drought resistance gene OsSINAT 5 and proteins encoded thereof and application, can improve the drought resistance of paddy rice.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: the application of a kind of paddy drought resistance gene OsSINAT 5 in cultivating the drought resistance paddy rice, the nucleotide sequence of this paddy drought resistance gene OsSINAT 5 is shown in SEQ ID NO:1.

Contain the application of recombinant expression vector in cultivating the drought resistance paddy rice of paddy drought resistance gene OsSINAT 5, described recombinant expression vector is divided into OsSINAT5 overexpression carrier and suppresses expression vector, this overexpression carrier is the recombinant expression vector that total length OsSINAT5 gene C DS SEQ ID NO:3 directed cloning is obtained under the pubix.nc 1300.ntap.gck carrier Ubiquitin promotor, and this inhibition expression vector is to utilize the pANDA carrier by OsSINAT5 genes encoding preceding 92 the inhibition expression vectors that amino acid whose DNA sequence obtain of PCR clone shown in SEQ ID NO:4.

A kind of method of cultivating the drought resisting paddy rice, be that the recombinant expression vector that will contain paddy drought resistance gene OsSINAT 5 imports in the rice cell, obtain the drought resisting paddy rice, this recombinant expression vector is divided into OsSINAT5 overexpression carrier and suppresses expression vector, this overexpression carrier is the recombinant expression vector that total length OsSINAT5 gene C DS SEQ ID NO:3 directed cloning is obtained under the pubix.nc1300.ntap.gck carrier Ubiquitin promotor, and this inhibition expression vector is the inhibition expression vector that utilizes the pANDA carrier to obtain by preceding 92 the amino acid whose dna sequence dnas of OsSINAT5 genes encoding of PCR clone shown in SEQ ID NO:4.

Utilize the proteins encoded of said gene:

1) utilize RNAi or other technology to suppress OsS INAT5 expression of gene, the present invention utilizes the pANDA carrier, makes up the RNAi expression vector by preceding 92 the amino acid whose dna sequence dnas of this rice Os SINAT5 genes encoding of PCR clone shown in SEQ ID No:4;

2), paddy drought resistance gene OsSINAT5 makes up the RNAi expression vector and imports plant tissue or cell, obtains transfer-gen plant.

The gene relevant provided by the present invention with paddy drought resistance, name is called OsSINAT5 (with SINAT5 homology in the Arabidopis thaliana), this gene is numbered Os03g24040 in rice genome database TIGR, the ubiquitin ligase that contains the RING type of " SINA " structural domain of encoding, the CDS total length 906bp of this gene, the albumen of coding 302aa.Up till now also without any research report about the OsSINAT5 function.Gene of the present invention promptly is the gene that is numbered Os03g24040 among the rice genome database TIGR, derives from paddy rice, is one of following nucleotide sequences:

1) dna sequence dna of sequence table SEQ ID No:1;

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

3) with sequence table in the dna sequence dna that limits of SEQ ID No:1 have the homology 90% or more and the identical function protein DNA sequence of encoding;

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

The rigorous condition of described height is in the solution of 0.1XSSPE (or 0.1XSSC), 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

The dna sequence dna of sequence 1 contains four exons by 4963 based compositions in the sequence table, be respectively from 5 ' the 601st at end is to the 799th bit base; From 5 ' the 2254th at end is to the 2477th bit base; From 5 ' the 2381st at end is to the 3216th bit base; From 5 ' the 3681st at end is to the 4363rd bit base.The nucleotide residue sequence of this sequence encoding SEQ ID No:2.

The proteins encoded OsSINAT5 of the gene OsSINAT5 relevant with rice drought tolerance, be protein, or the aminoacid sequence of SEQ ID No:2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residues and has identical active by SEQ ID No:2 deutero-protein with the amino acid residue sequence of SEQ ID No:2 with SEQ ID No:2 amino acid residue sequence in the sequence table.

The protein that sequence 2 amino acid residue sequences are made up of 302 amino-acid residues in the sequence table.

The expression vector, transgenic cell line and the host bacterium that contain gene OsSINAT5 of the present invention all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification OsSINAT5.

The carrier that sets out that is used to make up described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Utilize any one carrier that can guide foreign gene in plant, to express, OsSINAT5 gene of the present invention just performance drought of overexpression in plant is responsive, and with after this gene inhibition expression, plant shows as drought-enduring.

Gene of the present invention can add any one strong promoter or inducible promoter on being building up to plant expression vector the time before its transcription initiation Nucleotide, also can use enhanser.

Carry OsSINAT5 expression carrier of the present invention and can change vegetable cell over to, and cultivate the plant that drought resistance obtains improveing by using conventional biotechnological meanss such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection and electroporation.By the plant transformed host can be monocotyledonss such as paddy rice, corn, wheat, also is applicable to dicotyledonss such as tobacco, soybean, cultivates the plant variety of drought resisting.

Below in conjunction with the drawings and specific embodiments the present invention is described in further details.

Description of drawings:

Fig. 1 carries out the homology comparative result for adopting ClustalW software (public use software) with homologous protein in rice Os SINAT5 albumen and its Arabidopis thaliana.

Black is represented the amino acid of high conservative, " * "

Represent ring-finger and zinc-finger that it is conservative respectively.

Fig. 2-A and Fig. 2-B are the E3 ubiquitin ligase reaction of OsSINAT5 and OsSINAT5RING sudden change.Wherein:

Fig. 2-A is the synoptic diagram and the amino acid mutation site of OsSINAT5RING finger structural domain.

Fig. 2-B is the proteic external ubiquitin reaction of OsSINAT5.

At the E1 of external use wheat, people's E2 and 6xHis label ubiquitin (Ub), hatch altogether with the fusion rotein of GST-OsSINAT5 and GST-OsSINAT5 (H71Y) RING sudden change and to be that E3 is active to be detected.Left-hand digit shows proteic molecular weight, and unit is kilodalton (kD).RC represents common incubation time, and unit is minute (min).GST is the negative contrast of experiment.Sample is through the 8%SDS-PAGE electrophoretic separation, and Western detects the ubiquitin of His label with Nickel-HRP.

Fig. 3 is the Subcellular Localization of GFP-OsSINAT5 fusion rotein in rice protoplast.

18h locatees with laser co-focusing microscopic examination target protein after the transfection.From left to right be followed successively by among the last row figure: GFP is to impinging upon light field, green fluorescence down and light field and the green fluorescence result under merging; From left to right be followed successively by among the following row figure: the GFP-OsSINAT5 fusion rotein is under light field, the green fluorescence and light field and the green fluorescence result under merging.

Fig. 4-A is that OsSINAT5 overexpression transgenic plant Northern hybridization detects.

First road is contrast, and all the other are transgenosis independence transgenic line, and the application of sample amount of each sample is total RNA of 10ug, and 28SrRNA contrasts as the RNA application of sample.

Fig. 4-B is that OsSINAT5RANi transgenic plant RT-PCR detects.

First road is contrast, and all the other are transgenosis independence transgenic line, and the OsActin1 gene is used as confidential reference items.

Fig. 5-A to Fig. 5-D is the differential responses of OsSINAT5 transgenic plant to arid.

Carry out arid and handle experiment being incubated at 1/2MS substratum fortnight big plant, from left to right be followed successively by OsSINAT5 overexpression plant (OsSINAT5-OV), wild-type (WT), OsSINAT5RNAi suppresses plant (OsSINAT5-RNAi).

Fig. 5-A is that arid was handled after five days, OsSINAT5 transgenic plant and wild-type result relatively.

Fig. 5-B is that arid is handled after six days before the rehydration, the result of OsSINAT5 transgenic plant and wild-type comparison.

Fig. 5-C is after arid is handled six days rehydration 1d, the result of OsSINAT5 transgenic plant and wild-type comparison.

Fig. 5-D is that the OsSINAT5 transgenic plant were added up (n=15) with the wild-type surviving rate after arid was handled six days rehydration 1d.

Embodiment:

Experimental technique among the following embodiment if no special instructions, is ordinary method.

The acquisition of embodiment 1, plant adversity resistance related protein OsSINAT5 and encoding gene thereof

The result that little display (Microarray) is analyzed shows that rice Os 03g24040 gene is one and is subjected to drought-induced gene, utilize blast program find it with Arabidopis thaliana in SINAT5 homology highly, so called after OsSINAT5.Announced sequence according to paddy rice, the protein-coding region (SEQ ID No:3) of design primer amplification OsSINAT5 gene is used for further research.The clone PCR reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of 30sec, 59 ℃ of 30sec; 72 ℃ of 1min, 28 circulations; 72 ℃ are extended 10min.Auele Specific Primer is as follows:

F1 (5 '-GG

ATGGCCTCAGTTACTTATATTG-3 '), the italic base is the HindIII restriction enzyme site;

F2 (5 '-GG

TCACTGTTCCTTCCAAATTCTC-3 '), the italic base is the BamHI restriction enzyme site)

The fine total RNA reverse transcription of seedling of wild-type paddy rice Japan of extracting two all seedling ages obtains the first chain cDNA.With it is template, carries out pcr amplification with F1 and F2.The PCR product is carried out agarose gel electrophoresis detect, obtain the band that molecular weight is about 900kb.Reclaim test kit with sepharose and reclaim this fragment.Should reclaim fragment is connected with carrier pGEM-T Easy.To connect product transformed into escherichia coli DH5 α competent cell,, obtain containing the segmental recombinant plasmid of recovery according to the carboxylic Bian penicillin resistance label screening positive colony on the pGEM-T Easy carrier.With T7 on this recombinant plasmid vector and SP6 promoter sequence is that primer carries out nucleotide sequencing to it, sequencing result shows the protein-coding region of the OsSINAT5 gene that increased, the encoding histone region sequence of OsSINAT5 gene is shown in SEQ ID No:3, form the albumen shown in the coding SEQ ID No:2 by 906 deoxyribonucleotides.

The proteic ubiquitin ligase activity of embodiment 2, OsSINAT5

The method of employing vivoexpression OsSINAT5 albumen and RING finger mutain OsSINAT5 (H71) thereof is investigated it and whether is had the ubiquitin protein ligase activity.

The structure of 2,1 prokaryotic expression carrier pGEX-OsSINAT5

According to its Auele Specific Primer of sequences Design of OsSINAT5 gene, OsSINAT5 total length CDS directed cloning is gone in the pGEX-6p-1 fusion protein expression vector, change among the intestinal bacteria XL1-Blue with the thermal shock method.Expression of Fusion Protein, purifying are all undertaken by operation steps in the molecular cloning.Fusion rotein behind the purifying is used for the Ubiquitination reaction.The clone PCR reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of 30sec, 59 ℃ of 30sec; 72 ℃ of 1min, 28 circulations; 72 ℃ of extension 10min specific primer sequences are as follows

F1:5 '-GG

ATGGCCTCAGTTACTTATATTG-3 ', the italic base is the BamHI restriction enzyme site;

F2:5 '-GG

TCACTGTTCCTTCCAAATTCTC-3 ', italic base EcoRI is a restriction enzyme site)

2, the acquisition of 2OsSINAT5RING finger mutain

Shown in Fig. 1 and Fig. 2-A, there is conservative RING finger structural domain in OsSINAT5, in order to verify whether the proteic E3 ligase enzyme of OsSINAT5 is relevant with RING finger structural domain, we utilize quick rite-directed mutagenesis test kit (Stratagene) that the 211st cytosine(Cyt) C of OsSINAT5CDS sported VITAMIN B4 A, make reading frame sport AAT by CAT, shown in Fig. 2-A, coded amino acid becomes tyrosine (Y) by Histidine (H), has destroyed the conservative property of RING finger structural domain.After acquisition comprises the OsSINAT5CDS sequence of point mutation, carry out the structure of pGEX-OsSINAT5 (H71Y) according to OsSINAT5 construction of prokaryotic expression vector method in 2,1.The specific primer sequence of H71Y point mutation is as follows:

F1：5’-CATCAGTGCTCTAATGGTtATACATTGTGCTCTGGATGC-3’

F2:5 ' GCATCCAGAGCACAATGTAtaACCATTAGAGCACTGATG-3 ' 2, the external ubiquitin reaction of 3OsSINAT5 and OsSINAT5 (H71Y) mutain

Under the acting in conjunction that E3 (～1 μ g) that wheat (Triticum aestivum) E1 (GI:136632), people's E2 (UBCh5b) and 6xHis label ubiquitin (Ub), sublimed GST merge and ATP exist, His-Ub can be connected with GST-E3, and continuous increase along with His-Ub, form poly ubiquitin (Poly-Ub) chain, proteic molecular weight also increases thereupon.And whether albumen has with His-Ub and is connected, can press molecular weight size separation albumen by the SDS-PAGE gel electrophoresis after, test (selecting the pvdf membrane of 0.2 μ m for use) with the assorted His-Ub of Nickel-HRP through Western, analyze whether GST-E3 has added Poly-Ub.

Sublimed GST, GST-OsSINAT5, GST-OsSINAT5 (H71Y) albumen are added 1.5 μ L, 20 * reaction buffer (1M Tris pH7.5,40mM ATP sigma, 100mM MgCl2,40mMDTT), 3 μ L E1 (E1 that slightly carries, approximately 50ng), 3ul E2 (the E2 that slightly carries, about 40ng), the His-Ub of 2 μ L purifying (about 2 μ g), add ddH2O to cumulative volume be 30 μ L.On Eppendorf Thermomixercomfort instrument, 30 ℃, 900rpm is hatched, in order to verify further whether OsSINAT5 can add more Ub along with the increase of incubation time, we are provided with 15min to the OsSINAT5 wild-type protein, 30min, three incubation times of 60min, scarce E1, scarce E2, GST contrast and OsSINAT5 (H71Y) albumen test time are 90min.The every pipe in reaction back adds 10 μ l, 4 * SDS-PAGE sample-loading buffer, boils 5-10min in the boiling water, gets 15 μ L and carries out the SDS-PAGE electrophoresis.Carrying out Western hybridization behind the electrophoresis detects.As shown in Fig. 2-B, the GST-OsSINAT5 albumen of purifying is under the condition of hatching altogether with the E1 of wheat and people's E2, can detect OsSINAT5 and have the activity of external ubiquitinization, and, form increasing poly ubiquitin (Poly-Ub) chain along with the increase of incubation time 15min, 30min, 60min.Yet, OsSINAT5 (H71Y) albumen does not detect any poly ubiquitin chain yet after hatching 90min, the active completely dissolve of the E3 ligase enzyme of OsSINAT5 protein mutant OsSINAT5 (H71Y) illustrates that RING structure complete in the OsSINAT5 albumen is that OsSINAT5 albumen E3 ligase enzyme is active necessary really.

The proteic Subcellular Localization of embodiment 3, OsSINAT5

In order further to verify the proteic Subcellular Localization of OsSINAT5, the present invention is according to its Auele Specific Primer of sequences Design of OsSINAT5 gene, utilize pGDG (Goodin et al., 2002) vector construction the pGDG-OsSINAT5 expression vector, by rice protoplast transfection (Chen et al., 2006) make fusion gene moment expression in vegetable cell, 18h locatees with laser co-focusing microscopic examination target protein after the transfection.The clone PCR reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of 30sec, 59 ℃ of 30sec; 72 ℃ of 1min, 28 circulations; 72 ℃ of extension 10min Auele Specific Primers are as follows:

F1 (5 '-GG

ATGGCCTCAGTTACTTATATTG-3 '), the italic base is the HindIII restriction enzyme site;

F2 (5 '-GG

TCACTGTTCCTTCCAAATTCTC-3 '), the italic base is the BamHI restriction enzyme site);

Show with laser co-focusing microscopic examination moment expression of results, GSF is to impinging upon in the whole cell, comprise in tenuigenin and the nucleus all detecting, and the GFP-OsSINAT5 albumen overwhelming majority is distributed in nucleus, this result is consistent with its homologous protein SINAT5 in Arabidopis thaliana.

The structure and the conversion of embodiment 4, OsSINAT5 gene overexpression, inhibition expression vector

In order to illustrate the function of this gene better, the applicant suppresses it to express and overexpression in paddy rice, verify from the phenotype of transgenic plant.

OsSINAT5 gene overexpression and RNAi carrier adopt Gateway technology (Invitrogen).The overexpression carrier construction method is as follows: with total length OsSINAT5 gene C DS (SEQ ID No:3) directed cloning to pubix.nc1300.ntap.gck carrier (Puig at el., 2001) under the Ubiquitin promotor, be used to obtain paddy rice pUbiquitin-OsSINAT5 overexpression plant.It is as follows to suppress to express (RNAi) carrier construction method: find that by the comparison of Blast program the OsSINAT5 gene also has five homologous genes and has higher homology in paddy rice, there is tangible specificity (SEQ ID No:4) in preceding 92 amino acid whose dna sequence dnas but it is encoded, and select it as the target sequence that makes up the RNAi carrier.Clone this aim sequence by PCR, select pANDA carrier (Miki and Shimamoto, 2003) for use, recombining reaction is undertaken by the recombinant clone test kit specification sheets that Invitrogen company provides.

By agriculture bacillus mediated rice genetic method for transformation it is imported in the rice varieties " Japan fine ", through the callus of cultivating in advance, infecting, cultivating altogether, screening having hygromycin resistance, break up, take root, acclimatization and transplants, obtain transfer-gen plant.Agriculture bacillus mediated rice genetic method for transformation is used people's reported method (Qu et al., 2006) such as Qu.Briefly introduce as follows:

The substratum that 4,1 rice conversion relate to

1) callus inducing medium

NB:N6 macroelement and micro-ten B5 organic compositions, ten molysite, ten proline(Pro) 500mg/L, ten enzymic hydrolysis casein food grade (CEH) 300mg/L 10,4-D 2.5mg/L ten sucrose 30g/L ten plant gel 2.5g/L, pH5.8.

2) callus subculture medium

L3:L3 macroelement and micro-ten L3 organic compositions, ten molysite, ten proline(Pro) 500mg/L, ten glutamine 500mg/L 10,4-D 2.5mg/L ten maltose 30g/L ten plant gel 2.8g/L, pH5.8.

3) callus is total to culture medium

NS:N6 macroelement and micro-ten B5 organic composition+molysite, ten enzymic hydrolysis casein food grade (CEH) 1000mg/L, ten sucrose 30g/L, ten glucose 10g/L, ten Syringylethanone 200umol/L, ten plant gel 2.3g/L, pH5.2.

4) callus screening culture medium

S40:L3 macroelement and micro-ten L3 organic compositions, ten molysite, ten proline(Pro) 500mg/L, ten glutamine 500mg/L 10,4-D 2.5mg/L ten penbritin 200mg/L ten cephamycin 250mg/L ten maltose 30g/L ten Totomycin 40mg/L ten plant gel 3.0g/L, pH5.8.

5) callus breaks up substratum in advance

PDL:N6 macroelement and micro-ten B5 organic compositions, ten molysite, ten proline(Pro) 500mg/L, ten enzymic hydrolysis casein food grade ((CEH) 300mg/L ten 6-BA 1.0mg/L ten NAA2.0mg/L ten ABA5.0mg/L ten Totomycin 50mg/L ten sucrose 30g/L ten plant gel 3.0g/L, pH5.8.

6) callus division culture medium

DL:N6 macroelement and micro-ten B5 organic compositions, ten molysite, ten proline(Pro) 500mg/L, ten enzymic hydrolysis casein food grade ((CEH) 300mg/L ten 6-BA 2.0mg/L ten KT1.0mg/L ten NAA1.0mg/L ten IAA1.0mg/L ten maltose 30g/L ten plant gel 3.0g/L, pH5.8.

7) take root and the strong seedling culture base

1/2MS:1/2MS macroelement and micro-ten 1/2MS organic compositions, ten molysite, ten sucrose 30g/L, ten Totomycin 20mg/L, ten plant gel 3.0g/L, pH5.8.

8) CC substratum

CC macroelement and micro-ten CC organic compositions, ten molysite, ten proline(Pro) 500mg/L, ten glutamine 500mg/L, ten enzymic hydrolysis casein food grade (CEH) 300mg/L 10,4-D 2.5mg/L ten sucrose 30g/L ten plant gel 2.5g/L, pH5.8.

9) N6 substratum

N6 basal component ten proline(Pro) 500mg/L ten enzymic hydrolysis casein food grade (CEH) 300mg/L 10,4-D2.5mg/L ten sucrose 30g/L ten plant gel 2.5g/L, pH5.8.

10) YENB substratum

YE yeast powder extract 7.5g/L ten nutrient broth 8g/L ten agar powder 15g/L, pH7.0.

11) LB substratum

Peptone 10g/L ten yeast powder 5g/L ten NaCl 10g/L ten agar powder 15g/L, pH7.0.

12) SOB substratum

Peptone 20g/L ten yeast powder 5g/L ten NaCl 0.5g/L ten agar powder 15g/L, pH7.0.

13) SOC substratum

The 1mol/L Glucose Liquid of SOB substratum (sterilizing) ten 20ml degerming.

The competent preparation of 4,2 Agrobacteriums

1) Agrobacterium EHA105 bacterial strain list bacterium colony is inoculated in the 5mlYENB liquid nutrient medium, and 28 ℃, it is 0.4 that 200rpm is cultured to the OD600 value.

2) get Agrobacterium bacterium liquid 1ml, add in the 100ml YENB liquid nutrient medium in 1: 100 ratio, 28 ℃, 250rpm, shaking culture 2.5～3h.

3) nutrient solution is poured in the ice-cold aseptic centrifuge tube, put cooled on ice 10min, 4 ℃ of centrifugal 5min of 4000rpm abandon supernatant, use up raffinate as far as possible.

4) the resuspended precipitation of 0.1M CaCl2 of adding 34ml precooling is put 1h on ice.

5) 4 ℃, 4000rpm, centrifugal 5min abandons supernatant, flows to end raffinate as far as possible.

6) add 20% glycerine (precooling in the ice) 20ml, 4 ℃ of centrifugal 5min of 4000rpm abandon supernatant.

7) glycerine of adding 2ml 10% in precipitation, every pipe 80ul is sub-packed in the 1.5ml centrifuge tube, and-80 ℃ of refrigerators are preserved.

4,3 callus inductions and subculture

1) ripe rice paddy seed is shelled.

2) 70% alcohol disinfecting, 1～3min.

3) with 0.1% mercuric chloride sterilization 15min, constantly shake therebetween.

4) blot with aseptic washing 4～5 times, and with sterilization filter paper.

5) grain of rice is lain against 2,4-D concentration is on the callus inducing medium NB of 2.5mg/L, in 26 ℃ of dark 8～10d that cultivate, statistics callus induction rate.

6) callus is changed over to subculture among the subculture medium L3, carry out common cultivation behind 10～15d.

4,4 Agrobacteriums are to the conversion of rice callus tissue

1) Agrobacterium-mediated Transformation of picking-80 ℃ preservation is containing on the antibiotic LB solid medium line (50mg/L Kanamycin, 50mg/L Rifampicin), and 28 ℃ secretly are cultured to and grow bacterium colony.

2) picking list bacterium colony in 5mL contain in the antibiotic LB liquid nutrient medium (50mg/L Kanamycin, 50mg/LRifampicin), 28 ℃, 225rpm shaking culture 16～24h.

3) therefrom get 1ml bacterium liquid and join in 50mL AB (containing 50mg/L Kanamycin, the 50mg/L Rifampicin) liquid nutrient medium 28 ℃, 225rpm shaking culture, OD600=1.0.

4) room temperature, the centrifugal 20min of 4000rpm outwells liquid, collects thalline.

5) thalline is resuspended in the AAM liquid nutrient medium that 10ml is added with the 200umol/L Syringylethanone, 10 ℃, the centrifugal 10min of 4000rpm outwells liquid, collects thalline.

6) with containing the resuspended thalline of 200umol/L Syringylethanone AAM liquid nutrient medium, constant volume is to 50ml.

7) select fresh, faint yellow, fine and close, eugonic particulate state embryo callus of color and Agrobacterium bacterium liquid and mix according to a certain percentage, infect 30min.

8) after the suitable drying of callus, put into and be added with the 200umol/L Syringylethanone altogether on the culture medium, 26 ℃, secretly cultivate 3d.

The screening of 4,5 resistant callis and seedling differentiation

1) callus that is total to after cultivating uses rinsed with sterile water more than 10 times.

2) again with the sterilized water that contains 250mg/L cephamycin and the 200mg/L penbritin 1～2h that under 25 ℃, 150rpm condition, vibrates.

3) clean, suitably dry, to put on the screening culture medium of 40mg/L hygromycin selection pressure, 26 ℃ of dark cultivations behind 15～20d, are carried out second and are taken turns screening.

4) kanamycin-resistant callus tissue presorting earlier, 26 ℃, the dark cultivation; It is tender when white and hard to treat that resistant calli becomes, put into again in the division culture medium, and 26 ℃, illumination cultivation.

4,6 strong sprouts, domestication and transplanting

1) seedling that differentiation is obtained moves in the root media and cultivates.

2) regrowth grows to 15cm height and root and bottleneck is opened when comparatively sturdy, adds some sterilized waters and keep humidity to tame in culturing bottle.

When 3) growing new root, seedling is taken out, the substratum of root is carefully cleaned with flowing water, is connected in the soil of fetching from rice terrace and transplants.

4) for make after the transplanting young plant keep 85% humidity, adopt plastic film to cover, suitably ventilate behind the 2d, throw off film behind the 4d, carry out Routine Management.

The Molecular Detection of embodiment 5, OsSINAT5 gene overexpression, inhibition express transgenic plant

OsSINAT5 gene overexpression of the present invention, the total RNA of inhibition express transgenic plant extract the TRIzol method (Invitrogen) that adopts.OsSINAT5 gene overexpression transgenic plant Molecular Detection adopts Northern hybridization, and relevant experimental implementation method is undertaken by " molecular cloning ".The total RNA of 10 μ g behind the electrophoresis, is transferred on Hybond-N+ (Amersham) nylon membrane in denaturing formaldehyde glue.α- ³²The OsSINAT5 probe of P-dCTP mark is undertaken by random primer labelling test kit (Promega) specification sheets.Hybridization procedure reference Sambrook methods such as (1989) is carried out.28SrRNA is as the contrast of RNA application of sample, and Northern hybridization repeats twice at least.The result is shown in Fig. 4-A, and WT compares with contrast, and cloned genes OsSINAT5 of the present invention expression amount in most of transgenic lines increases, and can be used for further phenotypic evaluation.

The inhibition express transgenic Molecular Detection of OsSINAT5 gene adopts the RT-PCR method.The RNA sample is at first with DNaseI digestion, the pollution of removing genomic dna.Carrying out cDNA first chain according to the MMLV of Promega company (Cat.M170A) using method synthesizes: add following reactive component on ice successively: total RNA2 μ g, and 0.05 μ g Oligo-dT adds ddH2O to 12 μ L mixing, 75 ℃ of 5min, cooled on ice; Add following component more successively: 5 * reaction buffer, 5 μ L; RibonucLeose inhibitor 25U; DNTPs (10mM) 5 μ L, MMLV Reverse transcriptase (200U/ μ L) 1 μ L adds water to 25 μ L, 42 ℃ of reaction 1h, 70 ℃ of 10min termination reactions place on ice; With 10 times of synthetic strand cDNA dilutions, as the template of RT-PCR reaction.

The OsActin1 gene is used as confidential reference items, and reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of 30sec, 60 ℃ of 30sec; 72 ℃ of 35sec, 26 circulations; 72 ℃ are extended 10min.Its Auele Specific Primer is as follows:

F1(5’-CGTCTGCGATAATGGAACTGG-3’)

F2(5’-CTGCTGGAATGTGCTGAGAGAT-3’)

Select the OsSINAT5 specific fragment to detect, the PCR reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of 30sec, 59 ℃ of 30sec; 72 ℃ of 1min, 28 circulations; 72 ℃ are extended 10min.Auele Specific Primer is as follows:

F1(5’-GGAAGCTTATGGCCTCAGTTACTTATATTG-3’)；

F2(5’-GGGGATCCTCACTGTTCCTTCCAAATTCTC-3’)

The RT-PCR knot is shown in 4-B, and WT compares with contrast, and cloned genes OsSINAT5 of the present invention expression amount in most of transgenic lines descends, and can be used for further phenotypic evaluation.

The differential responses that embodiment 6, OsSINAT5 gene overexpression, inhibition express transgenic plant are handled arid

Under the prerequisite as a result of embodiment 5, the present invention has at first carried out the homozygote screening to OsSINAT5 gene overexpression, inhibition express transgenic plant T3 for plant.Concrete steps are as follows: each plant is chosen 30 seeds and shells, 70% ethanol surface sterilization 1min, and 30% sodium hypochlorite solution sterilization 30min, sterilization washing 5 times is containing the 1/2MS (Murashige﹠amp of Totomycin with the aseptic nipper sowing; Skong) the enterprising row filter of substratum plate, wild-type Japan is in contrast fine, and culture condition is 28 ℃, and continuous light intensity is 2500lux, 16h illumination/8h dark.Back statistics germination of two weeks and surviving rate, the plant that the present invention gives tacit consent to 100% surviving rate is a homozygote, each homozygote plant is taken a sample separately and carries out corresponding Molecular Detection.Get the Molecular Detection strain system consistent and be used for ensuing phenotypic evaluation with the hygromycin selection result.

The present invention chooses four, and independently overexpression and two independently suppress the express transgenic paddy rice of isozygotying and are used for phenotypic screen.Concrete steps are as follows: every pure and mild transgenic line selects 15 seeds to shell, 70% ethanol surface sterilization 1min, and 30% sodium hypochlorite solution sterilization 30min, sterilization washing 5 times is all filling 100ml 1/2MS (Murashige﹠amp with the aseptic nipper sowing; Skong) in the round shape culturing bottle of substratum, wild-type Japan is in contrast fine, and culture condition is 28 ℃, and continuous light intensity is 2500lux, 16h illumination/8h dark.Deng being cultured to for two weeks when big, OsSINAT5 overexpression plant (OsSINAT5-OV), wild-type (WT), OsSINAT5RNAi suppresses plant (OsSINAT5-RNAi) vegetative state does not have obvious difference.Open the culturing bottle lid, do not water and carry out the arid processing.

Arid is after five days, shown in Fig. 5-A, OsSINAT5 overexpression plant (OsSINAT5-OV) great majority wither and cover, blade is sagging, serious drought occurs and coerce performance, it is sagging that contrast wild-type (WT) also has partial blade to begin, and OsSINAT5RNAi suppresses plant (OsSINAT5-RNAi) and then keeping normal growth conditions substantially.

Arid is after six days before the rehydration, shown in Fig. 5-B, OsSINAT5 overexpression plant (OsSINAT5-OV) blade occurs expendable withered, most contrast wild-types (WT) and OsSINAT5RNAi suppress plant (OsSINAT5-RNAi) and have all withered all of a sudden, and part begins to occur expendable withered.

After the rehydration one day, shown in Fig. 5-C, OsSINAT5 overexpression plant (OsSINAT5-OV) blade can not recover fully, contrast wild-type (WT) has the partial blade state that restore normal growth, compare, most OsSINAT5RNAi suppress plant (OsSINAT5-RNAi) all can return to the normal growth state.The rehydration ability of plant after arid also is an important indicator of drought resisting, after rehydration, shown in Fig. 5-D, OsSINAT5RNAi suppresses plant (OsSINAT5-RNAi) survival rate the highest (54%), contrast wild-type survival rate is 27%, and all expendable the withering of OsSINAT5 overexpression plant (OsSINAT5-OV) covered, and can not recover growth.Arid and rehydration experiment show that the OsSINAT5 overexpression has reduced the drought tolerance of plant, have then strengthened the drought tolerance of plant after OsSINAT5 suppresses to express.These results have confirmed that OsSINAT5 has play a part in the response of plant to arid important.

Sequence table

＜110〉Agricultural University Of Hunan

＜120〉paddy drought resistance gene OsSINAT 5 and proteins encoded thereof and application

<130>PHW09012

<160>4

<170>PatentIn?version?3.1

<210>1

<211>4963

<212>DNA

<213>Oryza?sativa

<400>1

acattatttt?cgggagcaag?acatgaggag?ctcaaatcag?ttcctttccg?aacacagcac 60

acccctcctt?gacatgggcc?ggcccaaggc?ccagcacctg?tatctggtta?cggtatacta 120

agtctgcagt?taagtaatta?attaattatg?gtatgtgata?cttttctcaa?tacgtaataa 180

tgttcacttt?gatgttattt?tcatccatac?tatttttaga?taaagttacc?aaaaatttta 240

actaccttaa?gtttattgcc?aaattttagc?aagtaattat?gaaatcttgc?caaattttgg 300

caaccttgcc?aaaattttga?caacttagct?aaaatttggt?aatgccaaaa?cttgataagg 360

ttaaaaatgg?tagcaaagtg?aacaagcccg?aaatggtgtg?ttaaccactt?cggttaatta 420

atttcagtac?tcagttcatt?aaattaatta?aagaaaggcc?gaatccctag?aggtggggaa 480

cgggtgggcc?cgcgcaaacg?ccacgaggaa?gaaagagaga?gaaaaggtta?gcaactttca 540

cgggacaaaa?accagcagac?tagtcagata?tacatatact?cctcctcaac?aaaacaaaac 600

aaaaaacgcg?agaagagaga?gagagaagct?tttaattcga?attctctctc?tcatcacggc 660

ttcttctgct?tctccttctc?ctcccccctc?atccctcctc?cgccggtagc?cgccggccgg 720

gagcgagcag?cgagcgagac?cggagcccgc?cggagccggt?ctcgctcccc?cctcctccct 780

tgttcgcgcc?acccccgagg?tacatcccgc?cgatctctct?ctttctctct?ctcgttggtc 840

tgctctgtga?tttttttttt?ttttgccaag?gctgttcttt?atttggcaca?gagattgttt 900

tttttttcct?tctttgcggg?tgtccggttc?gagtccacgg?ggtttgactc?tgccggaaat 960

ggttcgaatg?cggcggccgg?agggcggagg?aggggttttg?gggggcgttc?ttggtgggtt 1020

ggtttgcgcc?gagtctttgt?taatttgtta?gggttcttga?tctgatgcgg?cttctcgttg 1080

ccgggatgcg?cttgcctcat?ggctgaattc?aaacaggccg?gtctctctct?ccctccctct 1140

ctcccgtcag?ccgtgtgctt?tgaccgagta?agtttttgaa?cttcgattgg?ttgagctggg 1200

ggttcttgac?tttttcttac?tccacgaatg?ttcctttttg?cacaatgggg?ctactaatga 1260

atggaaagct?agagatggtg?tgcctaatgg?tgatgtgccg?tcattgtgtt?agctcggatt 1320

tgtactgcga?atttgcgagc?tcacatgctc?agctgatgat?gtgttgatcc?agggagtata 1380

tataattggt?tggtagagta?cttgctagat?tgtgctcacg?ggtctcattt?caaagttcag 1440

tatgaaatct?tggttgtaaa?gtagtactag?tgttttgtta?atcgttaggg?agtacttctc 1500

ctgcattttt?cttaattttt?tccctttaaa?aactgtaggt?ggatttgtta?ttagtaaagc 1560

tgatttgtgc?caaatagaaa?ttctgtttta?tccgcagtaa?ggtactgtaa?acagtacttg 1620

caggcactag?ggtttattgg?caaggttttc?tatgttacat?tttaataaat?gccacctttt 1680

tcttcgtcaa?aagaagccac?ggttataaag?aaaaagtgaa?gcattaattg?atgtaaatgc 1740

acgaactaca?aatttccaca?aatataaacc?ttttaataaa?atccacaaat?gaaagaaaaa 1800

tgcagttcac?acaccaatct?ctttctcaaa?atttacactt?ttcttataac?aggtatgcta 1860

aattttctta?atacaaagca?tatacaaaac?atcacaagca?ctatagacat?tcatgacata 1920

tctgttgcat?aggacaatat?gcagtatgaa?gacctggctg?gtcaaaactt?ccgatgcaca 1980

tgtgaggccc?tatccatgtg?ctttcttgat?ttctgctcct?ttatggccct?atctccttca 2040

aagcccttaa?acacttcaac?agtgtccaat?catactatga?gactgtatgg?atatgggcat 2100

atggctatac?ttttgatgag?gcaatgattt?tttttgttaa?taatagtgtt?aataggaagt 2160

gttgaattgg?ttttttaaaa?ggaagtattt?aagcattgaa?tttgtcattt?attttgactc 2220

actaataaca?cagatagttc?tctttcctag?caggacccct?tacaatatct?agtgtattta 2280

tggcctcagt?tacttatatt?gatgactccg?gttctgaggt?aattgatcct?ccaaagactg 2340

aggtgctgga?tgttaccgaa?cttgctggtg?atcctgttcc?gcattcacca?aaaccaaatg 2400

tggtagtttc?tagcagtgtg?cgtgaactgc?ttgaatgtcc?agtctgcctg?agcgcaatgt 2460

atcctcccat?tcatcaggtg?cacatatacc?acttattgaa?tcttttttgc?acttaaaaag 2520

gatattttct?tgtatggaag?tcacttggga?aacaaaatgc?gtgtttcatt?taattgcatt 2580

gcttttacta?tcttgaatcc?tgctatcata?tttgtagtct?ggcctgatct?atttgaactt 2640

atctagatga?tttcctggtt?taaaacggtc?gttctgagct?gctttttcac?atatgttctg 2700

ttccactacc?aaattgttac?ccatgtattt?aagcaccaaa?aaagaagtta?gctaagatag 2760

tatgtacaat?atgcacattg?ttgtactatg?ttgtaagttg?taaccccaac?tctttttctg 2820

ctaatgcagt?gctctaatgg?tcatacattg?tgctctggat?gcaaaccaag?ggttcacaat 2880

cgctgtccaa?cttgtaggca?tgaactgggt?aatattagat?gtcttgctct?cgagaaggtg 2940

gctgcgtcgc?ttgaacttcc?atgcaagtac?cagaacttcg?ggtgtgtagg?catttatcct 3000

tactactgca?agctgaagca?tgagtcacag?tgccaatata?ggccttatag?ctgtccatat 3060

gctggatctg?aatgcacagt?tgctggtgac?attccatact?tggtgaatca?cttgaaagat 3120

gaccacaagg?ttgacatgca?taatggctgc?accttcaacc?atcgctatgt?caagtcgaat 3180

cctcatgaag?ttgagaatgc?cacctggatg?cttacggtat?cccctcgtta?actccaccat 3240

gttagttatt?gtgaagtata?tcactgatgt?ttacttatgc?aaagattggt?tcacttgtct 3300

gctaagatag?tccatatttc?tgtactgagt?tttatgagcc?tttctgtaaa?aataaacatt 3360

ggttcacttg?tacagtatga?ttgcactctg?tacttttcat?gtcaactgag?gatgctgatc 3420

atgcagcata?gttattggtc?taaaataatc?tctcacctat?gaagaaacta?tgcatcagaa 3480

tatccatctg?cttccatttc?caaaaaaaaa?aaaaaaaact?ttgtctgctt?tacagcacaa 3540

taatagcatt?tggacagggc?tcttgatctg?caaccttttc?ttgctgcttt?ctaagtgcaa 3600

tataaaatta?agaatttcct?tctaatctgt?tcttttccat?tgaacaaaat?ggactaagta 3660

actataatgc?ttccatctag?gttttcagct?gctttggcca?gtacttctgc?ctacatttcg 3720

aagcatttca?gctggggatg?gcacctgtgt?acatcgcctt?cctgaggttc?atgggtgatg 3780

acttggaagc?aaagaactac?agctacagcc?tggaggtagg?aggcactggc?cgcaaaatga 3840

tctggcaagg?ggttccccgg?agcatcagag?acagccatcg?gaaggtccgg?gatagctatg 3900

atgggcttat?catccaacgg?aacatggcct?tgttcttctc?tggtggagaa?aggaaggagc 3960

tcaaattgcg?ggtcactggg?agaatttgga?aggaacagtg?aaaataaaat?gtgaaatgat 4020

ctgttcatcg?ttcttaaccc?ttgcatgaac?ctatgtatca?tcacagtgcg?agaattatag 4080

ccattcatag?gcagtgactc?taaaatgaag?acttgtaatg?ttattagctt?gttttagcta 4140

ccatcttctg?ttagattggt?gcctgtggat?gactagatga?cagtcatgta?gaccagagtg 4200

gcactattgt?agaattcctg?gcaaacttct?ctttgccttg?aactgcttgt?tgagttgcca 4260

ttctgtggta?tagggaaatc?taatggcaat?tcatgagatg?aatgtgttga?actctttttc 4320

atgtttccag?catatttcta?gcttctttgt?tcattctttc?agcatatctc?tagcttgttt 4380

gttcattctt?tcagcatatt?tctagctatg?ttaaggcatg?atctgaaagt?tcttaagaat 4440

cggatatgct?gtaacaaatc?atggtgaaat?gtcatgtagc?atgtaccatt?tccttttatc 4500

ttaaatgctg?cttttgaatt?ggagggtgaa?taccagaatg?attttgtaag?ctttttccga 4560

ataaagtttc?aacttggtgt?gcaagattaa?atcggggttg?agcacccaga?gtaaatatta 4620

gtagaagata?aaatgaagtg?ctgatttttt?ttcttaaaga?aaaaacaaga?gttgaagcgc 4680

tagaatcaat?aatttgatct?gcatcatgtc?cggagtagta?ctgtttgtac?gctataggag 4740

caataatgtt?gtgcccgtac?gttactgcct?tcccttcaat?tgtctgcacc?tctgcacccg 4800

tcgtactact?gtgtcgtgga?cgtggcacct?tatctgaagg?atttcacaaa?atttgctcat 4860

ggaagtaagg?gttaagggag?tagtttgctt?tgatgcccag?atcaactcta?ccaaaatttg 4920

gtattgttaa?ctcaataagt?gtttttgatt?tttttttctt?tgc 4963

<210>2

<211>301

<212>PRT

<213>Oryza?sativa

<400>2

Met?Ala?Ser?Val?Thr?Tyr?Ile?Asp?Asp?Ser?Gly?Ser?Glu?Val?Ile?Asp

1 5 10 15

Pro?Pro?Lys?Thr?Glu?Val?Leu?Asp?Val?Thr?Glu?Leu?Ala?Gly?Asp?Pro

20 25 30

Val?Pro?His?Ser?Pro?Lys?Pro?Asn?Val?Val?Val?Ser?Ser?Ser?Val?Arg

35 40 45

Glu?Leu?Leu?Glu?Cys?Pro?Val?Cys?Leu?Ser?Ala?Met?Tyr?Pro?Pro?Ile

50 55 60

His?Gln?Cys?Ser?Asn?Gly?His?Thr?Leu?Cys?Ser?Gly?Cys?Lys?Pro?Arg

65 70 75 80

Val?His?Asn?Arg?Cys?Pro?Thr?Cys?Arg?His?Glu?Leu?Gly?Asn?Ile?Arg

85 90 95

Cys?Leu?Ala?Leu?Glu?Lys?Val?Ala?Ala?Ser?Leu?Glu?Leu?Pro?Cys?Lys

100 105 110

Tyr?Gln?Asn?Phe?Gly?Cys?Val?Gly?Ile?Tyr?Pro?Tyr?Tyr?Cys?Lys?Leu

115 120 125

Lys?His?Glu?Ser?Gln?Cys?Gln?Tyr?Arg?Pro?Tyr?Ser?Cys?Pro?Tyr?Ala

130 135 140

Gly?Ser?Glu?Cys?Thr?Val?Ala?Gly?Asp?Ile?Pro?Tyr?Leu?Val?Asn?His

145 150 155 160

Leu?Lys?Asp?Asp?His?Lys?Val?Asp?Met?His?Asn?Gly?Cys?Thr?Phe?Asn

165 170 175

His?Arg?Tyr?Val?Lys?Ser?Asn?Pro?His?Glu?Val?Glu?Asn?Ala?Thr?Trp

180 185 190

Met?Leu?Thr?Val?Phe?Ser?Cys?Phe?Gly?Gln?Tyr?Phe?Cys?Leu?His?Phe

195 200 205

Glu?Ala?Phe?Gln?Leu?Gly?Met?Ala?Pro?Val?Tyr?Ile?Ala?Phe?Leu?Arg

210 215 220

Phe?Met?Gly?Asp?Asp?Leu?Glu?Ala?Lys?Asn?Tyr?Ser?Tyr?Ser?Leu?Glu

225 230 235 240

Val?Gly?Gly?Thr?Gly?Arg?Lys?Met?Ile?Trp?Gln?Gly?Val?Pro?Arg?Ser

245 250 255

Ile?Arg?Asp?Ser?His?Arg?Lys?Val?Arg?Asp?Ser?Tyr?Asp?Gly?Leu?Ile

260 265 270

Ile?Gln?Arg?Asn?Met?Ala?Leu?Phe?Phe?Ser?Gly?Gly?Glu?Arg?Lys?Glu

275 280 285

Leu?Lys?Leu?Arg?Val?Thr?Gly?Arg?Ile?Trp?Lys?Glu?Gln

290 295 300

<210>3

<211>906

<212>DNA

<213>Oryza?sativa

<400>3

atggcctcag?ttacttatat?tgatgactcc?ggttctgagg?taattgatcc?tccaaagact 60

gaggtgctgg?atgttaccga?acttgctggt?gatcctgttc?cgcattcacc?aaaaccaaat 120

gtggtagttt?ctagcagtgt?gcgtgaactg?cttgaatgtc?cagtctgcct?gagcgcaatg 180

tatcctccca?ttcatcagtg?ctctaatggt?catacattgt?gctctggatg?caaaccaagg 240

gttcacaatc?gctgtccaac?ttgtaggcat?gaactgggta?atattagatg?tcttgctctc 300

gagaaggtgg?ctgcgtcgct?tgaacttcca?tgcaagtacc?agaacttcgg?gtgtgtaggc 360

atttatcctt?actactgcaa?gctgaagcat?gagtcacagt?gccaatatag?gccttatagc 420

tgtccatatg?ctggatctga?atgcacagtt?gctggtgaca?ttccatactt?ggtgaatcac 480

ttgaaagatg?accacaaggt?tgacatgcat?aatggctgca?ccttcaacca?tcgctatgtc 540

aagtcgaatc?ctcatgaagt?tgagaatgcc?acctggatgc?ttacggtttt?cagctgcttt 600

ggccagtact?tctgcctaca?tttcgaagca?tttcagctgg?ggatggcacc?tgtgtacatc 660

gccttcctga?ggttcatggg?tgatgacttg?gaagcaaaga?actacagcta?cagcctggag 720

gtaggaggca?ctggccgcaa?aatgatctgg?caaggggttc?cccggagcat?cagagacagc 780

catcggaagg?tccgggatag?ctatgatggg?cttatcatcc?aacggaacat?ggccttgttc 840

ttctctggtg?gagaaaggaa?ggagctcaaa?ttgcgggtca?ctgggagaat?ttggaaggaa 900

cagtga 906

<210>4

<211>276

<212>DNA

<213>Oryza?sativa

<400>4

atggcctcag?ttacttatat?tgatgactcc?ggttctgagg?taattgatcc tccaaagact 60

gaggtgctgg?atgttaccga?acttgctggt?gatcctgttc?cgcattcacc?aaaaccaaat 120

gtggtagttt?ctagcagtgt?gcgtgaactg?cttgaatgtc?cagtctgcct?gagcgcaatg 180

tatcctccca?ttcatcagtg?ctctaatggt?catacattgt?gctctggatg?caaaccaagg 240

gttcacaatc?gctgtccaac?ttgtaggcat?gaactg 276

Claims (3)

1. the application of paddy drought resistance gene OsSINAT 5 in cultivating the drought resistance paddy rice, the nucleotide sequence of this paddy drought resistance gene OsSINAT 5 is shown in SEQ ID NO:1.

2. contain the application of recombinant expression vector in cultivating the drought resistance paddy rice of paddy drought resistance gene OsSINAT 5, it is characterized in that: described recombinant expression vector is divided into OsSINAT5 overexpression carrier and suppresses expression vector, this overexpression carrier is the recombinant expression vector that total length OsSINAT5 gene C DS SEQ ID NO:3 directed cloning is obtained under the pubix.nc1300.ntap.gck carrier Ubiquitin promotor, and this inhibition expression vector is the inhibition expression vector that utilizes the pANDA carrier to obtain by preceding 92 the amino acid whose dna sequence dnas of OsSINAT5 genes encoding of PCR clone shown in SEQ ID NO:4.

3. method of cultivating the drought resisting paddy rice, be that the recombinant expression vector that will contain paddy drought resistance gene OsSINAT 5 imports in the rice cell, obtain the drought resisting paddy rice, this recombinant expression vector is divided into OsSINAT5 overexpression carrier and suppresses expression vector, this overexpression carrier is the recombinant expression vector that total length OsSINAT5 gene C DS SEQ ID NO:3 directed cloning is obtained under the pubix.nc1300.ntap.gck carrier Ubiquitin promotor, and this inhibition expression vector is the inhibition expression vector that utilizes the pANDA carrier to obtain by preceding 92 the amino acid whose dna sequence dnas of OsSINAT5 genes encoding of PCR clone shown in SEQ ID NO:4.

Images