CN1328383C - Wild rice drought-resisting gene and its coded protein and application - Google Patents

Abstract

The present invention discloses a drought-resisting gene of wild rice, a coding protein thereof and the application thereof. The gene can have one of the following nucleotide sequences: 1. the DNA sequence of SEQ ID No. 2 in a sequence table, and 2. a nucleotide sequence which can hybridize with a DNA sequence limited by the SEQ ID No. 2 in the sequence table under a high-strict condition. The coding protein of the gene has one of the following amino acid residue sequences: 1. SEQ ID No. 1 in the sequence table, and 2. a protein which causes the amino acid residue sequence of the SEQ ID No. 1 in the sequence table to be replaced, deleted or added by one to ten amino acid residues and has the functions of adjusting and controlling the drought resistance of a plant. The drought resisting gene provides foundation for manually controlling the expression of anti-stress genes and related stress resisting genes, and performs important functions in the cultivation of plants (especially paddy rice) with reinforced anti-stress performance and stress resistance.

Description

Anti-drought gene of wild-rice and proteins encoded and application

Technical field

The present invention relates in the plant to coerce relevant gene and proteins encoded and application, particularly anti-drought gene in the wild-rice and proteins encoded and its application in cultivating drought-resistant ability raising plant with anti-.

Background technology

The growth of plant is subjected to the influence of multiple abiotic factor in the environment, and wherein water stress is one of principal element that influences crop yield.Studies show that a large amount of adverse circumstance response genes be expressed as plant obtain resistance institute essential (Gonget al., PNAS, 99:11507-11512).Promoter region at water stress response genes such as most arids, high salt or freeze injuries all contains one or more dehydration reaction elements or makes C repeat (Dehydration-responsiveelement/C-repeat, be DRE/CRT), the core sequence of this element is G/ACCGAC (Shinozaki et al., Curr.Opin.Plant Biol., 3:217-223).DREB1/CBF (Dehydration-responsiveelement binding protein/C-repeat binding factor) transcription factor family by with this cis-acting elements in conjunction with expression (the Liu et al. that activates downstream gene, Plant Cell, 10:1391-1406).In recent years, in to the research of model plant Arabidopis thaliana, find constitutive expression bHLH (basicHelix-Loop-Helix) class transcription factor ICEl (inducer of CBF expressionl, ICEl) can with MYC recognition sequence in the CBF3 gene promoter area in conjunction with and regulate the DREBl/CBF gene transcription.ICEl can be modified (or combine with part) back and is activated, and causes the transcriptional expression of CBF and downstream gene thereof, thus the resistance (Chinnusamy et al., Genes Dev 17:1043-1054) of raising plant.

China Jiangxi Province Dongxiang common wild-rice (O.rufipogon) is the wild-rice kind that is distributed in the world northernmost, contain abundant disease-resistant worm gene and cold-resistant gene, have good resistance to cold, drought tolerance, anti-lean property and disease resistance etc., and protein content is higher, but utility value is huge.Therefore make full use of a series of desirable genes of Dongxiang Wild Rice, in plant breeding research, have great value.In addition, the finishing of Arabidopis thaliana and rice genome examining order is the research that utilizes these model plants the to carry out gene function condition of providing convenience.

Summary of the invention

The purpose of this invention is to provide anti-drought gene and proteins encoded thereof in the wild-rice.

Anti-drought gene provided by the present invention, name is called OrICLa, derives from common wild-rice (O.rufipogon), and it can have one of following nucleotide sequence:

1) dna sequence dna of SEQ ID No:2;

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

The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.101%SDS, hybridization and wash film under 65 ℃ of conditions.

SEQ ID No:2 in the sequence table is by 1706 based compositions, and its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID No:1 in the sequence table from 5 ' end 41-1615 bit base.

The albumen (OrICLa) that anti-drought gene of the present invention is coded, the protein with one of following amino acid residue sequences:

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

2) with the amino acid residue sequence of SEQ ID No:1 in the sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of regulation and control plant drought resistance.

SEQ ID No:1 in the sequence table is made up of 524 amino-acid residues.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and 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 OrICLa.

Utilize plant expression vector, anti-drought gene of the present invention is imported vegetable cell, can obtain transgenic cell line and transfer-gen plant arid tolerance enhanced.

Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprise the polyadenylic acid signal and any other participation mRNA processing or the dna fragmentation of genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein gene) 3 ' end to transcribe as the Agrobacterium crown-gall nodule all has similar functions.

When using OrICLa to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, root specific expression promoter 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, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, 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 OrICLa of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed cell or tissue is cultivated into plant.By the plant transformed host both can be monocotyledonss such as paddy rice, corn, also can be dicotyledonss such as Arabidopis thaliana.

Anti-drought gene OrICLa of the present invention will play an important role in cultivating resistance and resistance of reverse enhanced plant (particularly paddy rice) for the degeneration-resistant and anti-retrocorrelation expression of gene of artificial control provides the foundation.

The present invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 is the agarose gel electrophoresis detected result of the OrICLa of RT-PCR amplification

Fig. 2 is the physical map of OrICLa overexpression vector pSNICLa

Fig. 3 is the PCR qualification result of OrICLa transgenic arabidopsis foreign gene

Fig. 4 is the RT-PCR qualification result of OrICLa transgenic arabidopsis foreign gene

Fig. 5 is the phenotype of OrICLa overexpression Arabidopis thaliana after water stress is handled

Fig. 6 is the long statistics of OrICLa overexpression Arabidopis thaliana root after water stress is handled

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

The clone of embodiment 1, OrICLa

Amino acid residue sequence " GKKKGMPAKNLMAERRRRKKLNDRLYMLRSVVPKISKMDRASILGDAIDYLKELLQ RINDLHNELES " according to ICEl bHLH structural domain, in ncbi database, retrieve (blastp), find two full length cDNA sequences that homology is higher in rice genome, according to the following primer of nucleotide sequence design of one of them gene (OrICLa): 5 ' holds primer: CGC GGATCCCATCTCCTTCCCCACCCC (band underscore part base is a restriction enzyme BamH I recognition site); 3 ' end primer: CGG GGTACCGCCCTGAGCAGGTCTAAAACTA (band underscore part base is a restriction enzyme Kpn I recognition site).Is template with Dongxiang Wild Rice (O.rufipogon) seedling through 4 ℃ of total RNA that handled 30 minutes, under the right guiding of above-mentioned primer, and the cDNA sequence of RT-PCR amplification OrICLa, concrete grammar may further comprise the steps:

1, the extraction of total RNA: the Dongxiang Wild Rice seedling in two weeks of growth on not containing the 1/2MS substratum of hormone is placed in the low temperature incubator 4 ℃ of processing 30 minutes, extract the total RNA of seedling with Trizol method (agents useful for same is available from Invitrogen company), concrete grammar is: collect the rice material 100mg through 4 ℃ of subzero treatment, place liquid nitrogen to grind immediately, add 1mL Trizol reagent, fully behind the mixing, room temperature was placed 5 minutes; Add the 0.2mL chloroform, violent jolting 15 seconds, room temperature incubation 3 minutes; 4 ℃, centrifugal 15 minutes of 12000g; Supernatant liquor is transferred in the new 1.5mL centrifuge tube, added 0.5mL isopropanol precipitating RNA; At last with RNA precipitation with being dissolved in after 1mL 75% washing with alcohol in right amount in the DEPC treated water ,-70 ℃ of preservations are standby.

2, the first chain cDNA's is synthetic: use Superscript ^TMII RT test kit (Invitrogen) is also operated by the test kit specification sheets: get the total RNA of paddy rice that 1-5 μ g step 1 the obtains PCR pipe of RNase of having put into deactivation, add Oligo (dT) _12-18(500mg/mL) μ L and dNTP Mix (10mM each) 1 μ L is supplemented to 12 μ L with the distilled water after the DEPC processing, heats 5 minutes down at 65 ℃ behind the mixing, places on ice 1 minute then rapidly.Of short duration 5 * the first chains synthetic damping fluid 4 μ L, 0.1M DTT 2 μ L and the RNaseOut of adding again after centrifugal ^TM(40unit/ μ L) 1 μ L, gently behind the mixing, 42 ℃ of incubations 2 minutes add Superscript then ^TMII ThermoScript II (200unit/ μ L) 1 μ L, mixing, 42 ℃ of incubations 50 minutes, 70 ℃ of heating made enzyme deactivation in 15 minutes, obtained the first chain cDNA.

3, OrICLa cDNA's is synthetic: the reverse transcription product of getting the acquisition of 1 μ L step 2 is a template, under the guiding of 5 ' end primer and 3 ' end primer, with the cDNA of the synthetic OrICLa of the method for PCR, the PCR reaction system is: LA Taq (TaKaRa company) 0.5 μ L, 2 * GC damping fluid (TaKaRa company), 25 μ L, dNTP 1 μ L, 5 ' end primer (10 μ M/L), 1 μ L, 3 ' end primer (10 μ M/L), 1 μ L, template 1 μ L, add distilled water postreaction system to 50 μ L.The PCR reaction conditions is: earlier 94 ℃ 4 minutes; Again 94 ℃ 45 seconds, 62 ℃ 45 seconds, 72 ℃ 2 minutes, totally 35 circulations; Last 72 ℃ 10 minutes.

After reaction finishes, the PCR product is carried out 0.8% agarose gel electrophoresis detect, detected result is (swimming lane M is Marker, and swimming lane 1 is the RT-PCR product of OrICLa) as shown in Figure 1, obtains the band that molecular weight is about 1.8kb, conforms to expected results.Reclaim test kit (sky, Beijing is a Time Inc.) with sepharose and reclaim this fragment, should reclaim fragment then and be connected with carrier pGEM-T Easy (Promega), linked system is: T ₄Dna ligase (3u/ μ L), 2 * ligase enzyme damping fluid, 5 μ L, pGEM-T Easy (50ng/ μ L) 0.5 μ L and recovery PCR product 3.5 μ L, 4 ℃ were reacted 12-24 hour.Method (Proc Natl Acad Sci with reference to Cohen etc., 69:2110), to connect product transformed into escherichia coli DH5 α competent cell, according to the carboxylic Bian penicillin resistance label screening positive colony on the pGEM-T Easy carrier, obtain containing the segmental recombinant plasmid of recovery, called after pTE-OrICLa.With T7 on this plasmid vector and SP6 promoter sequence is that primer carries out nucleotide sequencing to it, sequencing result shows that OrICLa has the nucleotide sequence of SEQ ID No:2 in the sequence table, by 1706 based compositions, its its open reading frame (ORF) is that coding has the protein of the amino acid residue sequence of SEQ ID No:1 in the sequence table from 5 ' end 41-1615 bit base; From 5 ' end 1022-1222 bit base is the encoding sequence of ICE1 bHLH conserved domain, 67 amino acid of encoding.Carry out homology relatively with ICE1, the homology of Nucleotide and aminoacid sequence is respectively 42% and 40%.

The structure of embodiment 2, OrICLa overexpression vector pSNICLa

1, the acquisition of corn ubiquitin promoter (UbiPro)

1) extraction of corn gene group DNA: the about 0.2g corn seedling of clip places liquid nitrogen to grind; Add the extraction damping fluid (containing 0.1M Tris-HCl pH8.0,50mM EDTA, 0.5M NaCl, 1%SDS and 1% beta-mercaptoethanol) of the new preparation of 800 μ L then, thermal agitation makes its whole suspensions; 65 ℃ of water-baths 30 minutes were put upside down mixing once in per 5 minutes; Add the 5M potassium acetate of 250 μ L precoolings then, put upside down mixing immediately, ice bath 5 minutes; Add equal amounts of phenolic/chloroform, extracting once, centrifugal 5 minutes of 12000rpm; Collect supernatant, add the isopropanol precipitating DNA of 0.6 times of volume, room temperature was placed 40 minutes; Centrifugal 15 minutes of 4 ℃ of 12000rpm abandon supernatant; Precipitation is respectively washed once with 70%, 100% ethanol; After the drying, be dissolved in the ddH that 20 μ L contain 100 μ g/mL RNase ₂Among the O, obtain corn gene group DNA.

2) pcr amplification corn ubiquitin promoter (UbiPro): the corn gene group DNA solution of getting the acquisition of 2 μ L step 1) is having 5 ' primer (GG of HindIII recognition site as template AAGCTTCTGCAGTGCAGCGTGACCCGG) and have 3 ' primer (CG of BamHI recognition site GGATCCAAGTAACACCAAACAACAGGG) carry out pcr amplification under the guiding, the PCR reaction conditions is: earlier 94 ℃ 3 minutes; Again 94 ℃ 45 seconds, 62 ℃ 45 seconds, 72 ℃ 2 minutes, totally 35 circulations, last 72 ℃ 10 minutes.After reaction finishes, the PCR product is carried out 0.8% agarose gel electrophoresis to be detected, show and obtain the amplified fragments that length is about 2kb, conform to expected results, reclaim this purpose fragment, with reclaiming behind restriction enzyme Hind III and the BamH I double digestion, obtain having the corn ubiquitin promoter (UbiPro) of sticky end, standby.

2, with restriction enzyme Sac I and EcoR I Noster poly A terminator sequence is downcut from plasmid vector pBI 221 (Clontech company), be connected in the corresponding site of carrier pUC19 (TaKaRa company), obtain recombinant vectors, called after pUC19-Noster.Use restriction enzyme HindIII and BamHI double digestion pUC19-Noster again, after agarose gel electrophoresis detects, reclaim the big fragment of linearizing carrier, and will reclaim the corn ubiquitin promoter (UbiPro) that has sticky end that fragment and step 1 obtain and link to each other, obtain recombinant vectors, called after pUN19.

3, partially digested and HindIII complete degestion is purchased the recombinant vectors pUN19 that builds from step 2 and is downcut the fragment that the length that comprises UbiPro and Noster is about 2.3kb with restriction enzyme EcoR I, this fragment cloning is gone into plasmid vector pCAMBIA1301 (Center for the Application of Molecular Biology toInternational Agriclture, www.cambia.org) the EcoR I of multiple clone site and HindIII site, obtain recombinant vectors, called after pUN1301.

4, with restriction enzyme HindIII and BamHI plasmid vector pBI221 is carried out double digestion, after detecting, reclaims agarose gel electrophoresis the 35S promoter fragment that length is about 0.8kb, it is connected with plasmid vector pUN1301 through the same enzyme double digestion, obtain containing the segmental recombinant vectors of 35S promoter, called after pSN1301.

5, recombinant plasmid vector pTE-OrICLa and pSN1301 are carried out double digestion with restriction enzyme KpnI and BamHI respectively, the enzyme system of cutting is: plasmid 5 μ L, 10 * enzyme cutting buffering liquid, 2.5 μ L, KpnI 1 μ L, BamHI 0.8 μ L add ddH ₂O postreaction system to 50 μ L, 37 ℃ of enzymes were cut 8 hours.With agarose electrophoresis enzyme is cut product and separate, reclaim the OsICLa fragment of 1.7Kb and the big fragment of carrier pSN1301 of 13Kb, be dissolved in 45 μ L ddH respectively ₂Among the O.By following reaction system both are connected again: T ₄The OsICla 10 μ L of dna ligase 2 μ L, 10 * ligase enzyme damping fluid, 2 μ L, recovery, pSNl301 6 μ L, 16 ℃ connect 16 hours.To connect product transformed into escherichia coli DH5 α competent cell, and obtain positive strain through the Kan+ plate screening, with this recombinant plasmid called after pSNICLa, its physical map as shown in Figure 2.Adopt CaMV 35S strong promoter to start goal gene OrICLa overexpression in plant in this plasmid, the preparation method of transgenic plant is stated embodiment as follows.

The acquisition and the evaluation thereof of embodiment 3, OrICLa overexpression Arabidopis thaliana

The Agrobacterium that contains expression vector is soaked seed that plants transformed is tied and uses T by the plant that this seed grows up to ₀Representative, T ₁T is shown in representative ₀The seed that produces for selfing reaches by the plant that it grew up to T ₂T is shown in representative ₁The seed that produces for selfing and by plant that it grew up to.

One, transforms the acquisition of the Arabidopis thaliana that OrICLa overexpression plasmid pSNICLa is arranged

1, OrICLa overexpression plasmid pSNICLa arabidopsis thaliana transformation

Operate with EasyJecT Plus electric exciter (Britain EquiBio company) and with reference to specification sheets, plasmid pSNICLa is transformed Agrobacterium C58 with electrization, the resistant panel screening through containing kantlex obtains the positive colony of Agrobacterium; Refer again to the method (Clough SJ and Bent AF, 1998 Plant J 16:735-43) of Clough etc., under the mediation of above-mentioned positive colony Agrobacterium, with the pSNICLa arabidopsis thaliana transformation.

2, the antibiotic screening of the positive seedling of transgenic arabidopsis

The seed of the transgenic arabidopsis that the step 1 of results is obtained soaked 10 minutes with 0.2%TritonX-100; Use 10% clorox surface sterilization again, 12 minutes; Aqua sterilisa washing five times, per 2 minutes are once; Water is layered on seed on the MS flat board that contains the 25mg/L Totomycin, with masking foil parcel, places 2 days under 4 ℃, dark condition, takes out back dark cultivation 3-4 days in 23 ℃ culturing room; After 3-4 days, the positive for just being sieved to that growing way is the highest transforms seedling, takes out under the lucifuge condition, cultivates under the light 3 days again, the transgenic positive seedling is moved to cultivate in the flowerpot then, obtains T1 for transgenic line.

Two, the evaluation of transgenic arabidopsis

GUS staining fluid: 100mmol/L phosphoric acid salt pH 7.O, 0.1%Triton X-100,10mmol/L EDTA, the 0.5mmol/L Tripotassium iron hexacyanide, X-Gluc 1mg/mL.

Edwards extracts damping fluid: 200mM Tris-Cl pH7.5,250mM NaCl, 25mM EDTA, 0.5%SDS.

1, the GUS dyeing of the positive seedling of Arabidopis thaliana is identified

The positive seedling leaves of the Arabidopis thaliana tip 2-3mm material of getting step 1 screening in 3 weeks of growth carries out GUS dyeing.37 ℃ of incubations 12 hours, with the decolouring of 75% alcohol, blade is blue positive plant again.

2, the PCR of transfer-gen plant identifies

1) extraction of genomic dna

Get a slice and put into 1.5mL Eppendorf pipe, add liquid nitrogen, grind pestle with tissue and ground for 10 seconds, grind with the blade of the Arabidopis thaliana positive plant of the method evaluation of step l; Add 400 μ L Edwards and extract damping fluid, grind gently (organizing on the flush away pestle); Vibrated for 5 seconds, centrifugal 1 minute, the supernatant liquor that shifts 300 μ L added 300 μ L Virahols in new pipe, mixed, and placed 2 minutes in room temperature; Centrifugal, abandon supernatant, air-dry precipitation is dissolved in the 100 μ L water, obtains the genomic dna of transgenic line.

2) PCR of transfer-gen plant identifies

The genomic dna that obtains with step 1) is a template, primer 1 (5 ' end primer): CGCGGATCCCATCTCCTTCCCCACCCC and primer 2 (3 ' end primer): under the guiding of CGGGATCCAAGTAACACCAAACAACAGGG, method with PCR identifies that to transfer-gen plant the PCR reaction system is: each 1 μ L of genomic dna solution 1 μ L, LA taqO.5 μ L, 2 * GC damping fluid, 25 μ L, dNTP 1 μ L, primer 1 (10 μ M/L) and primer 2 (10 μ M/L), add distilled water postreaction system to 50 μ L.The PCR reaction conditions is: earlier 94 ℃ 4 minutes; Again 94 ℃ 45 seconds, 62 ℃ 45 seconds, 72 ℃ 2 minutes, totally 35 circulations; Last 72 ℃ 10 minutes.After reaction finishes, the PCR product is carried out 0.8% agarose gel electrophoresis to be detected, the result is (swimming lane Marker is the DNA standard molecular weight) as shown in Figure 3, swimming lane 6,8,10,11 and 15 is different transgenic lines, swimming lane WT is the wild-type plant), all transgenic lines all expand the positive band that about 1.8kb, conform to the expection size, wild-type does not then have, and shows that goal gene successfully changes in the Arabidopis thaliana.

3, the RT-PCR of transfer-gen plant identifies

Respectively the used WT of extraction step 2 and total RNA in five transgenic line flowering period respectively get the total RNA of 2 μ g and carry out reverse transcription, synthetic its cDNA, and RNA extracts and reverse transcription method carries out with reference to the step among the embodiment 1.With this reverse transcription product is template, at OrICLa special primer (5 ' primer: GTGCCCAAGATCAGCAAGATGGACAG, 3 ' primer: under guiding GGTACCTGTCGAAAATGCCACATGACC), carry out RT-PCR and detect (is with Actin with reference to), the PCR reaction system is: rTaq 0.2 μ L, dNTP 2 μ L, 10 X PCR damping fluids, 2 μ L, (10 μ M) 5 ' primer 0.4 μ L, (10 μ M) 3 ' primer 0.4 μ L, 20 X cDNA, 2 μ L, add ddH ₂O postreaction system to 20 μ L.The PCR reaction conditions is: earlier 94 ℃ 4 minutes; Again 94 ℃ 45 seconds, 59 ℃ 45 seconds, 72 ℃ 90 seconds, totally 35 circulations; Last 72 ℃ 10 minutes.After reaction finishes, the PCR product is carried out 0.8% agarose gel electrophoresis to be detected, (swimming lane 6,8,10,11 and 15 is different transgenic lines to the result as shown in Figure 4, swimming lane WT is the wild-type plant), all transgenic lines all expand and the fragment of OrICLa clearly, wild-type does not then have, and shows that OrICLa successfully changes in the Arabidopis thaliana.

The phenotype of the OrICLa transgenic arabidopsis after embodiment 4, water stress are handled

To not have the OrICLa transgenic arabidopsis seedling that sprouted 2 days on the hormone culture-medium at normal MS transfers to and carries out lack of water on the MS substratum that contains 300mM N.F,USP MANNITOL and coerce processing, grow after 10 days, observe phenotype and its root length is added up, the phenotype observations is (A: do not have the wild-type plant of coercing as shown in Figure 5; B: do not have the transfer-gen plant of coercing; C: the wild-type plant that coerced by lack of water; D: the transfer-gen plant that coerced by lack of water), to the long statistics of root as shown in Figure 6, above-mentioned test-results shows that the OrICLa transgenic arabidopsis has tangible water stress resistance.

Sequence table

<160>2

<210>1

<211>524

<212>PRT

＜213〉common wild-rice (O.rufipogon)

<400>1

Met?Leu?Pro?Arg?Phe?His?Gly?Ala?Met?Trp?Met?Gln?Asp?Asp?Gly?Gly

1 5 10 15

Gly?Asp?Gln?Glu?His?Gly?Gln?Ala?Ala?Pro?Pro?Gly?Gln?Glu?Gln?His

20 25 30

His?His?Asp?Gln?His?Leu?Met?Ala?Leu?Ala?Ala?Ala?Ala?Ala?Gly?Gly

35 40 45

Ala?Gly?Phe?Gly?Ala?Ala?Gln?Ala?Pro?Ala?Pro?Leu?Leu?Asp?Glu?Asp

50 55 60

Trp?Tyr?Phe?Asp?Ala?Ala?Gly?Gly?Gly?Gly?Gly?Gly?Ala?His?Gly?Ser

65 70 75 80

Met?Met?Leu?Gly?Leu?Ser?Ser?Val?His?Gly?Gly?Ile?Gly?Ala?Gly?Thr

85 90 95

Ser?Gly?Gly?Gly?His?Gly?Gln?Gln?Phe?Ser?Leu?Leu?Asn?Met?Gly?Ala

100 105 110

Ala?Ala?Ala?Pro?Phe?Asp?Val?Ser?Gly?Phe?Asp?Leu?Gly?Ile?Ala?Cys

115 120 125

Gly?Gly?Val?Gly?Gly?Gly?Gly?Asp?Val?Val?Ser?Phe?Leu?Gly?Gly?Gly

130 135 140

Asn?Ala?Ser?Asn?Thr?Ala?Leu?Leu?Pro?Val?Gly?Ash?Ala?Gly?Phe?Leu

145 150 155 160

Gly?Thr?Phe?Gly?Gly?Phe?Gly?Thr?Ala?Ala?Ser?Gln?Thr?Pro?Glu?Phe

165 170 175

Gly?Gly?Leu?Ala?Gly?Phe?Asp?Met?Phe?Asp?Ala?Gly?Ala?Val?Asn?Thr

180 185 190

Gly?Gly?Ser?Ser?Ser?Ser?Ser?Ser?Ala?Ala?Ala?Ala?Ala?Ala?Ser?Ala

195 200 205

Ser?Ala?His?Val?Ser?Asn?Thr?Ala?Pro?Phe?Ser?Gly?Arg?Gly?Lys?Ala

210 215 220

Ala?Val?Leu?Arg?Pro?Leu?Asp?Ile?Val?Pro?Pro?Val?Gly?Ala?Gln?Pro

225 230 235 240

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

245 250 255

Asp?Asp?Lys?Lys?Arg?Lys?Ala?Ala?Ala?Gly?Ala?Gly?Ala?Gly?Ala?Leu

260 265 270

Ser?Ala?Asp?Gly?Ala?Asp?Met?Val?Leu?Asp?Asp?Gly?Asp?Asp?Asp?Gly

275 280 285

Leu?Ser?Ile?Asp?Ala?Ser?Gly?Gly?Leu?Asn?Tyr?Asp?Ser?Glu?Asp?Ala

290 295 300

Arg?Gly?Gly?Glu?Asp?Ser?Gly?Ala?Lys?Lys?Glu?Ser?Asn?Ala?Asn?Ser

305 310 315 320

Thr?Val?Thr?Gly?Asp?Gly?Lys?Gly?Lys?Lys?Lys?Gly?Met?Pro?Ala?Lys

325 330 335

Asn?Leu?Met?Ala?Glu?Arg?Arg?Arg?Arg?Lys?Lys?Leu?Asn?Asp?Arg?Leu

340 345 350

Tyr?Met?Leu?Arg?Ser?Val?Val?Pro?Lys?Ile?Ser?Lys?Met?Asp?Arg?Ala

355 360 365

Ser?Ile?Leu?Gly?Asp?Ala?Ile?Glu?Tyr?Leu?Lys?Glu?Leu?Leu?Gln?Lys

370 375 380

Ile?Asn?Asp?Leu?Gln?Asn?Glu?Leu?Glu?Ser?Ser?Pro?Ala?Thr?Ser?Ser

385 390 395 400

Leu?Pro?Pro?Thr?Pro?Thr?Ser?Phe?His?Pro?Leu?Thr?Pro?Thr?Leu?Pro

405 410 415

Thr?Leu?Pro?Ser?Arg?Ile?Lys?Glu?Glu?Ile?Cys?Pro?Ser?Ala?Leu?Pro

420 425 430

Ser?Pro?Thr?Gly?Gln?Gln?Pro?Arg?Val?Glu?Val?Arg?Leu?Arg?Glu?Gly

435 440 445

Arg?Ala?Val?Asn?lle?His?Met?Phe?Cys?Ala?Arg?Arg?Pro?Gly?Leu?Leu

450 455 460

Leu?Ser?Ala?Met?Arg?Ala?Val?Glu?Gly?Leu?Gly?Leu?Asp?Val?Gln?Gln

465 470 475 480

Ala?Val?Ile?Ser?Cys?Phe?Asn?Gly?Phe?Thr?Leu?Asp?Ile?Phe?Lys?Ala

485 490 495

Glu?Gln?Cys?Lys?Asp?Gly?Pro?Gly?Leu?Leu?Pro?Glu?Glu?lle?Lys?Ala

500 505 510

Val?Leu?Met?Gln?Ser?Ala?Gly?Leu?His?Thr?Met?Ile

515 520

<210>2

<211>1706

<212>DNA

＜213〉common wild-rice (O.rufipogon)

<400>2

ccatctcctt?ccccacccca?ccgccattgc?cgccgcggcg?atgctgccgc?ggtttcacgg 60

cgccatgtgg?atgcaggacg?acggcggcgg?cgaccaagaa?cacgggcagg?cggcgccgcc 120

tgggcaggag?cagcaccacc?acgaccagca?tctcatggcg?ttggcggccg?cggccgcggg 180

cggcgccggg?ttcggcgcgg?cgcaggcgcc?ggcgccgctg?ctcgatgagg?actggtactt 240

cgacgcggcg?ggtggtggtg?gtggtggcgc?gcatgggtcc?atgatgctgg?gtttgtcgtc 300

cgtccatggc?gggattgggg?cggggacgtc?tggtggtggg?catgggcagc?agttctcgct 360

gctcaacatg?ggcgccgcgg?ccgcgccgtt?cgacgtctcc?gggttcgacc?tcgggatcgc 420

ctgcggcggc?gttggcggcg?gcggcgacgt?ggtgtcgttt?cttggcggcg?ggaacgcgtc 480

gaacaccgcg?ctgctccccg?tcgggaacgc?ggggttcctc?ggcacgttcg?gcgggttcgg 540

caccgcggcg?tcccaaacgc?cggagttcgg?cgggctcgcc?gggttcgaca?tgttcgacgc 600

gggcgccgtg?aacaccgggg?gcagctcctc?ctcctcgtcg?gcggcggcgg?cggcggcgtc 660

cgcctcggcg?cacgtgagca?acaccgcgcc?gttctccggg?cgcggcaagg?cggcggtgct 720

gcggccgctg?gatatcgtcc?cgcccgtggg?cgcgcagccg?acgctgttcc?agaagcgcgc 780

gctccgccgc?aacgccggcg?aggacgacga?cgacaagaag?cgcaaggccg?ccgcgggcgc 840

gggcgcgggc?gcgctgtccg?ccgacggcgc?cgacatggtg?ctcgacgacg?gcgacgacga 900

cggcctcagc?atcgacgcgt?cgggcggcct?caactacgac?tccgaggacg?ccaggggcgg 960

cgaggacagc?ggcgccaaga?aggagtcgaa?cgccaacagc?acggtcaccg?gcgacgggaa 1020

ggggaagaag?aaggggatgc?cggccaagaa?cctcatggcg?gagcgccgcc?gccggaagaa 1080

gctcaacgac?cgcctctaca?tgctccgctc?cgtcgtgccc?aagatcagca?agatggacag 1140

ggcttccatt?ctcggcgacg?cgattgagta?cctgaaggag?ctgctgcaga?agatcaatga 1200

tcttcagaat?gagctcgagt?cgtcccccgc?gacgtcgtca?ttgcctccaa?cacccacaag 1260

cttccatccc?ctgacaccga?cgctgcccac?attgccgtcc?cgcatcaagg?aagagatctg 1320

cccaagtgca?ttgccaagcc?ccactggaca?acagccaagg?gttgaggtta?ggctgaggga 1380

aggccgggct?gtcaatatcc?acatgttctg?tgctcggagg?cccggtctac?tgctctctgc 1440

catgagggcc?gtcgaaggcc?ttggtctcga?tgtccagcaa?gctgtaatca?gttgcttcaa 1500

tggctttacg?ttggatattt?ttaaggctga?gcaatgcaag?gacggccctg?ggctgttgcc 1560

tgaagaaatc?aaggccgttc?tgatgcaatc?cgccgggctc?cataccatga?tctaggacag 1620

gagagctcaa?tcaaactcca?aaggacagag?tagctcagga?attgacaaag?taccggtgtt 1680

tcctggtcat?gtggcatttt?cgacag 1706

Claims (7)

1, wild-rice anti-drought gene, its base sequence is shown in SEQ ID NO:2.

2, the described anti-drought gene encoded protein of claim 1, its amino acid residue sequence is shown in SEQ ID NO:1.

3, contain the described expression carrier of claim 1.

4, the transgenic cell line that contains the described gene of claim 1.

5, the host bacterium that contains the described gene of claim 1.

6, a kind of method of cultivating drought-resistant plant is to utilize plant expression vector that the described anti-drought gene of claim 1 is imported vegetable cell, obtains arid tolerance enhanced transgenic cell line and transfer-gen plant.

7, method according to claim 6 is characterized in that: described is paddy rice by the plant transformed host.

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