CN101870983B - Wintersweet gene containing thioesterase functional domain and application in drought-tolerant gene engineering thereof - Google Patents

Abstract

The invention relates to a wintersweet gene containing a thioesterase functional domain and application in drought-tolerant gene engineering thereof, belonging to the field of molecular biology and gene engineering. The invention provides the wintersweet gene containing the thioesterase functional domain and a protein and meanwhile provides an expression vector and a host cell which correspond to the gene. The invention has the advantages that the application of the wintersweet gene containing the thioesterase functional domain can improve the drought tolerance of a transgenic plant.

Description

The wintersweet gene and the anti-drought gene practical applications thereof that comprise the thioesterase domain

Technical field

The invention belongs to molecular biology and genetically engineered field, be specifically related to contain the wintersweet gene clone and the application thereof of thioesterase domain.

Background technology

In plant materials, synthetic protokaryon approach and the eucaryon approach of comprising of the lipid acid of plastid.Lipid acid de novo synthesis in the plastid can be stopped by the plastid acyltransferase, and carboxyl groups is gone to the glycerolipid of plastid from acyl-ACP, and this is the protokaryon approach; Perhaps,, discharge free fatty acids and ACP, then lipid acid is exported plastid, delivered in the coenzyme A pond by coenzyme A esterification again with the acyl-ACP hydrolysis by the acyl-ACP thioesterase.Source as the endoplasmic reticulum glycerolipid goes out, and this is the eucaryon approach.Thioesterase belongs to esterase family esterase, is a kind of of lytic enzyme.Can the acyl-ACP hydrolysis be discharged free fatty acids and ACP.

The biosynthesizing of lipid acid mainly occurs in the matrix of plastid, through series reaction, generates different chain length (the fatty acyl ACP of C8～C18) by acetyl-CoA.The final step reaction that in plastid, takes place is that thioesterase discharges free lipid acid from ACP, so in the lipid acid de novo synthesis, the acyl-ACP thioesterase is played an important role for the termination of carbochain.

Relatively different according to amino acid whose sequence with the specificity of enzyme substrates, be divided into the fatty acyl-acp thioesterase of two kinds of base types in the plant.One type of called after FatA is for 18:1 ^{Δ 9}-ACP activity is the highest, is determining that 18:1 outputs to the outer level of plastid in the plant materials so infer FatA.Second type is representative with the 16:0-ACP thioesterase, the highest to the saturated acyl-acp activity than short chain.FatB is 16:0-ACP＞18:1 to the catalytic activity of substrate ^{Δ 9}-ACP＞18:0-ACP＞14:0-ACP.Because acyl group ACP thioesterase has substrate specificity,, be the chain length of decision lipid acid and the principal element of sfas level so its activity influence the ratio between the fatty acid component in the various glycerolipids.

Discover that recently the FatB gene is ancient in plant materials, exist in each organ of plant that expression amount is the highest in spending.Infer that separating of FatA and FatB offspring is more Zao than the separation of camphor tree plant, estimate to separate before 1.5-3.0 is 100000000 years that FatA is by the FatB deutero-.The medium-chain thioesterase is in the angiosperm evolutionary process, is come through evolutionary process differentiation several times by the 16:0 thioesterase.

Thioesterase has the domain of acyltransferase, can acyl group be separated from acyl group-acyl carrier protein.

Acyl-ACP thioesterase catalysis FAS round-robin stops, purifying from various plants, and encoding sox is also cloned from various plants such as safflower, rape and mouseearcress.Different acyls-ACP needs different enzymes, and is special to oleoyl-ACP like the thioesterase of cloning in the safflower, and the thioesterase of cloning in the Arabidopis thaliana is to 14～18 carbon saturation of substrates tool specificitys.

Acyl group ACP thioesterase becomes in the lipid biosynthetic enzyme system first through transforming, and changes transgenic plant over to produce the enzyme of economic product.Chain acyl ACP thioesterase during the work of the Calgene of biotech company has at first proved and existed, and biochemical evidence is provided.From California bay, cloned the 12:0-ACP thioesterase then.The gene transferred plant of the single middle chain acyl ACP thioesterase of coding can obviously be changed the length of lipid acid stored in the seed oil.Being specific to the lauric acyl group ACP thioesterase in bay, the thioesterase that is specific to other chain lengths has also been identified from different plants.Other kind of medium chain fatty acid is rich in other productions, also obtains like myristic acid and the sour transgenic plant of certain herbaceous plants with big flowers.

When research arabidopsis mutant body (Fatb-ko), find that the growth velocity of two mutants is lower than the wild-type Arabidopis thaliana, after 4 weeks of growing, the two mutants fresh weight is compared with wild-type plant and is reduced 50%.And the seed development ability of two mutants is low, and morphology changes.In two mutants; In different tissues; The total quantity of sfas reduces 40%-50% than wild-type; This minimizing occurs over just in the wax layer, the triacyl glycerol in the seed on fatty acid component in the cytosol, leaf and stem surface, and finds that two mutants all is affected at plant strain growth and seed germination.

Wintersweet is a kind of well-known how anti-plant, the extremely strong anti-low temperature that it had, drought-resistant, resistant to diseases and insects, and to contain a large amount of wax layers relevant with the blade surface of wintersweet for this, and wax layer is to be made up of longer chain fatty acid.In numerous research, also do not see the relevant relevant report of wintersweet gene aspect the degeneration-resistant physiological function of plant that contains the thioesterase domain.

Summary of the invention

(1) a kind of dna sequence dna is provided, its a kind of wintersweet gene that contains the thioesterase domain of encoding, called after CpAcylTase; (2) provide a kind of gene engineering method of utilizing to obtain the CpAcylTase encoded protein; (3) application of CpAcylTase gene aspect plant drought is provided.

The invention provides the CpAcylTase gene, it has the nucleotide sequence shown in SEQ ID NO:1 in the sequence table.

From wintersweet corolla cDNA library; Utilize the PCR method screening and cloning to go out the CpAcylTase gene: to utilize RNAiso Reagent (available from Dalian Takara company) to extract the total RNA of wintersweet corolla; According to the synthetic wintersweet corolla cDNA library of SMARTTM cDNA Library ConstructionKit; 1000 library clone order-checkings of picking and bioinformatic analysis obtain wintersweet corolla resistance ESTs sequence at random.From Wintersweet Flower ESTs sequence, find the cDNA sequence of coding CpAcylTase, according to this sequences Design upstream and downstream primer, and be that template is carried out pcr amplification with Wintersweet Flower cDNA library, clone the full-length gene of CpAcylTase.

The gene of CpAcylTase contains a complete open reading frame by 1110 based compositions, is the SEQ ID NO:1 sequence in the sequence table.The initiator codon of opening code-reading frame is ATG, and terminator codon is TAG.

The present invention also provides the albumen that relates to the CpAcylTase genes encoding, and it has the aminoacid sequence shown in the SEQ ID NO:2 in the sequence table, is to contain 369 amino-acid residues, and its theoretical molecular size is 41.7kDa, and the prediction iso-electric point is 7.72.According to preceding 18 left and right sides amino acid of psort analysis CpAcylTase albumen is signal peptide, and this albumen is stabilize proteins.

Recombinant prokaryotic expression vector contains the CpAcylTase gene, and the recombinant prokaryotic expression vector transformed host cells is intestinal bacteria.

The recombinant plant expression vector contains the CpAcylTase gene, and recombinant plant expression vector transformed host cells is an agrobacterium tumefaciens, and the genetically modified host cell that the recombinant plant expression vector transforms is a vegetable cell.

Of embodiment two, engineered method also capable of using gives expression to the CpAcylTase albumen of biologically active in efficient escherichia expression system.

About the CpAcylTase gene in the application aspect the plant drought:

The CpAcylTase gene comprises with the expression vector transformed plant cells that makes up in the application of cultivating on the resistance plant, the plant transformed cell culture is become plant.The CpAcylTase gene coding region is cloned among the plant expression vector pETV7, it is imported tobacco leaf, obtain the tobacco strain system of overexpression, improve the ability of changeing CpAcylTase genetic tobacco (abbreviation transgene tobacco) drought resisting through the Agrobacterium infestation method.

In that Nicotiana gossei and transgene tobacco are carried out water-retentivity experiment discovery, change the loss that the CpAcylTase genetic tobacco can greatly reduce moisture, the percentage of water loss behind its stomatal closure is compared Nicotiana gossei and has been reduced by 30%.

Pass through drought stress; The drought-resistant ability of finding transgene tobacco is apparently higher than Nicotiana gossei; At drought stress after 10 days; The serious wilting of wild-type tobacco reaches 100%, and serious wilting of transgene tobacco is less than 2%, explains that the relative Nicotiana gossei of transgene tobacco has shown stronger drought-resistant ability of coercing damage.

Beneficial effect of the present invention is to provide a kind of CpAcylTase gene and albumen, has made up plant expression vector, and obtains transgenic plant, and the CpAcylTase gene can improve the drought-resistant ability of transgenic plant.

Description of drawings

Fig. 1 is for being template with wintersweet corolla cDNA, the pcr amplification electrophorogram

Wherein: M:marker size from top to bottom is: 1850bp, 1470bp, 1090bp, 740bp, 420bp, 280bp

1: the purpose band

Fig. 2 is a SDS-PAGE prokaryotic expression whole protein electrophorogram

Wherein: M:Marker is respectively 66,43,31 (KDa)

1,2,3: the BL21 host bacterium whole protein that contains recombinant clone plasmid pET-28a::CpAcylTase

CK: the BL21 host bacterium whole protein that contains empty carrier plasmid pET-28a

Fig. 3 is the building process synoptic diagram of CpAcylTase plant expression vector

Fig. 4 is that the positive seedling RT-PCR of transgene tobacco identifies electrophorogram

Wherein: M:marker size from top to bottom is: 2000bp, 1000bp, 750bp, 500bp, 250bpLine-1, Line-2, Line-3, Line-4: the positive seedling RT-PCR of transgene tobacco W: wild-type tobacco RT-PCR

Fig. 5 is that drought stress compares synoptic diagram to the influence of transgene tobacco and Nicotiana gossei

Wherein: W: Nicotiana gossei T: transgene tobacco

Fig. 6 is a relatively synoptic diagram of Nicotiana gossei, the analysis of transgene tobacco water-retentivity

Embodiment

Embodiment one: clone CpAcylTase gene

1. extract RNA:

Get 500mg wintersweet corolla and extract the total RNA of Wintersweet Flower with RNAiso Reagent.

2. construction cDNA library:

Get total RNA, according to the synthetic Wintersweet Flower cDNA library of SMARTTM cDNA Library Construction Kit, 1000 library clone order-checkings of picking and bioinformatic analysis obtain Wintersweet Flower resistance ESTs sequence at random.

3. clone the clone of CpAcylTase gene fragment:

According to the cDNA sequence of the coding CpAcylTase that finds out in the Wintersweet Flower ESTs sequence, design upstream and downstream primer, and be that template is carried out pcr amplification with Wintersweet Flower cDNA library.

Auele Specific Primer is following:

CpAcylTase-P1:5 '-CCC

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

CpAcylTase-P2:5 '-CCG

CTAATGCGATATGGAATTAGGGT-' 3, the italic base is the XhoI restriction enzyme site.

The clone PCR reaction conditions is: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 50sec; 54 ℃ of annealing 30sec; 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 10min.Amplified production detects through 1% agarose gel electrophoresis, and a specific band (Fig. 1) is arranged on the 1000bp position.And then by ordinary method with fragment cloning in pMD18-T Vector (purchase white Dalian Takara company), and through Shanghai bio-engineering corporation checks order.

The CpAcylTase Gene Sequence Analysis

Comprise 1110bp through this gene cDNA sequence ORFs of order-checking, contain a complete open reading frame, be the SEQ ID NO:1 sequence in the sequence table.369 amino acid of encoding have the aminoacid sequence shown in the SEQ ID NO:2 in the sequence table, and the molecular weight size is 41.7kDa, and the prediction iso-electric point is 7.72.The result shows the cDNA sequence that has obtained full-length gene.

Utilize NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) that the amino acid of this genes encoding is carried out the conservative region analysis, the result shows: this albumen belongs to the acyl-ACP thioesterase.

Aminoacid sequence to the CpAcylTase genes encoding on the NCBI website carries out BLAST; Carry out evolutionary analysis and homology analysis: CpAcylTase with other species aminoacid sequences of confirming function and Semen Myristicae acyl-ACP thioesterase amino acids coding evolutionary distance is nearest, with Arabidopis thaliana amino acid sequence homology of the same race be 48%.

According to preceding 18 left and right sides amino acid of psort analysis CpAcylTase is signal peptide, is stabilize proteins.With SOPMA CpAcylTase is carried out secondary structure prediction, find to contain 39.84% α spiral (h), 5.42% βZhuan Jiao (t), 14.09% extended chain (e), 40.65% curl at random (c).

Embodiment two: CpAcylTase efficiently expressing in intestinal bacteria

1. construction of prokaryotic expression vector:

In order to show the encoding function of CpAcylTase gene, the CpAcylTase gene clone is arrived between the HindIII and BamH I site of pET-28a (+), and transformed into escherichia coli BL21, obtain to efficiently express.

Design and synthesize a pair of Auele Specific Primer:

5 '-CGC CTAGTAATGAGCATGATT-' 3 (the italic base is the restriction enzyme site of BamHI)

5 '-CCC

GTAGTTCCTTATGCGATA-' 3 (the italic base is the restriction enzyme site of HindIII)

Press molecular cloning normal experiment program, amplify the gene coded sequence that two ends have specific restriction enzyme site with the method for PCR, gene is connected with carrier pMD18-T, checking is correct.Get pMD18::CpAcylTase plasmid and pET-28a (+) plasmid (purchasing ancient cooking vessel state biotech firm) respectively with BamHI, HindIII double digestion, be connected again in Shanghai; To connect in the product transformed into escherichia coli BL21 bacterial strain (purchasing biotech firm) in Kai Ji, and with the LB plate screening recon that contains the 100ug/ml kantlex.After plasmid enzyme restriction evaluation and PCR evaluation, the recon that the connection of sifting out is correct checks order, and proves that the reading frame of the dna sequence dna that inserts carrier is correct.Have the CpAcylTase expression carrier being built into, name pET-28a::CpAcylTase is used for the abduction delivering analysis.

2. prokaryotic expression:

The bacterial strain that will contain the pET-28a::CpAcylTase recon is inoculated in LB substratum (1L: peptone 10g, yeast extract 5g, NaCL10 gram), and 37 ℃ are cultured to bacterium liquid OD ₆₀₀=0.4-0.6.The recombinant expressed bacterium of BL21 is ground and ultrasonication, and we have obtained containing the BL21 host bacterium crude enzyme liquid of recombinant clone plasmid pET-28a::CpAcylTase.Carry out the 12%SDS-PAGE electrophoresis detection, as can be seen from Figure 2, the CpAcylTase gene efficiently expresses in BL21.

Embodiment three: contain the structure of the plant expression vector of CpAcylTase gene, and the preparation of transgene tobacco

CpAcylTase gene coding region of the present invention is cloned into plant expression vector pTEV7, and (Jilin University molecular biology of plants research department preserves; Yin Junhua, clone and the functional study of Wintersweet Flower aquaporin CpTIP cDNA, middle National IP Network includes; 2008.) in; Through the Agrobacterium infestation method it is imported in the tobacco, make the tobacco strain system of overexpression, improve the tobacco drought-resistant ability.

1. the structure of plant expression vector

Design a pair of primer, introduce the restriction enzyme site of HindIII and XhoI respectively,

CpAcylTase-P1:5 '-CCC

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

CpAcylTase-P2:5 '-CCG

CTAATGCGATATGGAATTAGGGT-' 3, the italic base is the XhoI restriction enzyme site.

Press molecular cloning normal experiment program, amplify the purpose fragment with the method for PCR, with fragment cloning to pMD18-T Vector, order-checking.To identify that correct positive recombinant plasmid pMDl8::CpAcylTase and plant expression vector pETV7 carries out HindIII and XhoI double digestion through order-checking.And enzyme is cut product reclaim, connect, be built into purpose carrier pTEV7::CpAcylTase (building process is seen Fig. 3) and be transformed among the agrobacterium tumefaciens EHA105.

2. utilize leaf dish infestation method transformation of tobacco

(1) picking is inoculated in the liquid nutrient medium through the Agrobacterium of identifying, 28 ℃, the about 48h of 250rpm shaking culture are to the logarithmic growth later stage; Centrifugal, abandon supernatant; Thalline is with 1/2MS liquid medium (1/2 * MS macroelement, 1 * MS trace element, 1 * MS VITAMINs; 1 * molysite, pH5.7-5.8) suspension is diluted to about OD600=0.5;

(2) get aseptic tobacco leaf, remove the main lobe arteries and veins, it is cut into small pieces;

(3) tobacco leaf that shears is placed MS division culture medium (1 * MS macroelement, 1 * MS trace element, 1 * MS VITAMINs; 1 * molysite, sucrose 30g/L, 6-BA 3mg/L; NAA 0.2mg/L, agar powder 8g/L, pH5.7-5.8); 28 ℃, light application time 16h/ days, intensity of illumination 2000LX cultivated 2 days;

(4) tobacco leaf that will cultivate 2 days immerses 10min in the said firm ready bacterium liquid of the first step, on aseptic filter paper, blots bacterium liquid, blade is placed on the MS substratum 28 ℃ of dark cultivations 2 days;

(5) blade that is total to after cultivating washs 3 times with the sterilized water that contains cephamycin 300mg/L earlier after microcolony occurring around it; Wash 1 time with the MS nutrient solution that contains cephamycin 300mg/L again; Blot with aseptic filter paper then; Change on the MS screening culture medium that contains kantlex constant temperature culture (condition is with cultivating in advance), per 15 days replacing one subcultures over to;

When (6) treating that bud grows to the 1cm left and right sides, downcut and move in the root media, short its taken root.After treating root system development, number, and move into and fill in the flowerpot of sterile soil, preserved moisture 2 days the room temperature Routine Management with plastics film transforming seedling.

With RT-PCR method validation transgene tobacco

1. from the tobacco leaf that has imported the CpAcylTase gene, extract total RNA with ordinary method; With the negative contrast of Nicotiana gossei; The result shows that 28S and 18S strip-type are neat, and the 28S band is obviously bright in the 18S band, explains that total RNA integrity is fine; Do not degrade, can identify as further reverse transcription.With the tobacco RNA of the commentaries on classics CpAcylTase gene that extracts is that primer obtains strand cDNA through reverse transcription with CpAcylTase-P2.

2. being template again with cDNA, is that primer carries out pcr amplification with CpAcylTase-P1, CpAcylTase-P2 then.Amplified production detects through 1% agarose gel electrophoresis; One specific band is arranged on the corresponding position; Consistent with CpAcylTase Gene Partial clip size, and on the swimming lane of wild-type plant negative control this band not, explain that the transfer-gen plant that detects is positive.This has proved that tentatively the CpAcylTase gene has been integrated in the tobacco gene group, and on rna level, has obtained expression (like Fig. 4).

Embodiment four: drought stress is to the influence of transgene tobacco and Nicotiana gossei

Choose full wild and transgene tobacco seed and sow in soil in (vermiculite: the vegetation charcoal is 1: 1) 25 ℃, light application time 16h/ days, intensity of illumination 2000LX.After two weeks, select the consistent seedling of growth, be transplanted in the basin.After recovering 2 weeks of growth, choose the consistent healthy plant of upgrowth situation its drought stress of cutting off the water is jointly handled, the discovery wild-type tobacco is wilted and is reached at 100% o'clock after 8 days, and wilting situation (like Fig. 5) does not but appear in transgene tobacco.

Embodiment five: the rate-of-loss of coolant of excised leaf is measured

It is close to get under the equal culture condition size, and Nicotiana gossei identical in quality and transgene tobacco blade are put into 28 ℃ of incubators after taking by weighing fresh weight.For the blade pore is closed fully, after 150min, begin to measure moisture and reduce situation, the quality that after this every separated 1h takes by weighing blade is measured 5 times altogether, calculates the percentage of water loss (like Fig. 6) of two kinds of blades:

Percentage of water loss=(weight after fresh weight-dehydration)/fresh weight

The interior relative Nicotiana gossei of percentage of water loss of transgene tobacco unit time had reduced by 30% after the result showed stomatal closure.

Sequence table

The sequence of SEQ ID NO.1

(i) sequence signature: (A) length: 1110bp; (B) type: Nucleotide; (C) chain property: strand.

(ii) molecule type: Nucleotide

(iii) sequence description: SEQ ID NO.1

1 ATGAGCATGA?TTGCCAGCAG?TGTAGGTGCT?GCTTTTTTTC?CAGCCCAAGG?CATCATCAAG

61 TCCAAGCCGG?CTGGGTTGCA?CGTGAAAGCA?AATGGCCGAG?CCTCCCCCAG?CATTGACGGT

121 CCGAAGGTGA?CCGTCGGCCT?AGAGGGCACG?AACGCCTCGT?CCACAAGGAA?ATTCATGAAC

181 TTGTTGCCTG?ACTGGAGCAT?GCTACTTGCC?GCCTTTACAA?CCATCTTTGA?GAAGCAGAAG

241 GTTGTGGTCG?ACCAGTTTCG?ATTCGGCCAT?GACAGGCTGG?TTTACAGTGA?GAATTTCACA

301 ATAAGGTCAT?ATGAGATAGG?TGCTGATCAG?ACGGCATCAA?TAGAGACAGT?GATGAATCTT

361 TTGCAGGAGA?CTGGAATCAA?CTGTTTTAGG?AGCCTTGGGC?TTTTACTTGA?TGGTTTTGAT

421 TCAACAGTGG?AGATGTGTAA?GAGAGATCTT?ATATGGGTTG?TGACTCGTAT?GCAGGTTATC

481 GTTGATCACT?ATCCTTCTAG?GGGTGATACT?GTTGAAGTAG?AGACACACTG?CGGTGCATAT

541 GGAAAGCATG?GCCACCGCCG?AGAATGGCTA?ATCCGGAACA?GCAAAACTGG?TCAAATTCTT

601 ACACGAGCTA?CCAGTGTTCT?GGTGGTGATG?AATAAGCGGA?CGAGGAGATT?GTCCATATTA

661 CCTGATGAAG?TTAGAAGGGA?ATTAGAGCCT?TATTTCATGG?AGAATCTTAG?TGTGATGAAG

721 GACCAAGGCA?GAAAACTTCC?CAAGGTCGAT?CATAGCATTG?CAGATTACGT?CCGACAAGGG

781 TTGACTTGTC?AATGGAGTGA?TTTGGATATC?AATCAGCATG?TAAACCATAT?CAAATACGTT

841 AAATGGATTT?TTGAGAGTGT?TCCGGTTTCT?ATCTTAGAAA?GTCACGAGAT?TTCCAGCATG

901 ACTCTTGAAT?TTAAGAGAGA?GTGCGGCAAG?GATAGCATGT?TGCAGTCTCT?GACTGCCGTC

961 GTGTCCGGTC?GCAGGGTTGA?CGGATCAGTA?GAAGAAACTG?ACGTTGAATT?TCAGCACTTG

1021?CTCCAGCTTG?AAGATGGGCC?TGAGGTCATG?AGGGGAACAA?CAAAGTGGAG?ACCCAAGAGT

1081?ACCCTGTTCC?CTAATTCCAT?ATCGCA TTAG

The sequence of SEQ ID NO.2

(i) sequence signature: (A) length: 369 amino acid; (B) type: amino acid; (C) chain property: strand.

(ii) molecule type: polypeptide

(iii) sequence description: SEQ ID NO.2

1 MSMIASSVGA?AFFPAQGIIK?SKPAGLHVKA?NGRASPSIDG

41 PKVTVGLEGT?NASSTRKFMN?LLPDWSMLLA?AFTTIFEKQK

81 VVVDQFRFGH?DRLVYSENFT?IRSYEIGADQ?TASIETVMNL

121 LQETGINCFR?SLGLLLDGFD?STVEMCKRDL?IWVVTRMQVI

161 VDHYPSRGDT?VEVETHCGAY?GKHGHRREWL?IRNSKTGQIL

201 TRATSVLVVM?NKRTRRLSIL?PDEVRRELEP?YFMENLSVMK

241 DQGRKLPKVD?HSIADYVRQG?LTCQWSDLDI?NQHVNHIKYV

281 KWIFESVPVS?ILESHEISSM?TLEFKRECGK?DSMLQSLTAV

321 VSGRRVDGSV?EETDVEFQHL?LQLEDGPEVM?RGTTKWRPKS

361 TLFPNSISH*

Claims (7)

1. a wintersweet gene that comprises the thioesterase domain is characterized in that it is the nucleotide sequence shown in the SEQ ID NO:1 in the sequence table.

2. albumen is characterized in that it is by the aminoacid sequence shown in the SEQ ID NO:2 in the described wintersweet gene order coding that comprises the thioesterase domain of claim 1, the sequence table.

3. a recombinant prokaryotic expression vector is characterized in that containing the wintersweet gene that comprises the thioesterase domain as claimed in claim 1.

4. a recombinant plant expression vector is characterized in that containing the wintersweet gene that comprises the thioesterase domain as claimed in claim 1.

5. recombinant prokaryotic expression vector transformed host cells with claim 3, described host cell is intestinal bacteria.

6. recombinant plant expression vector transformed host cells with claim 4, described host cell is an agrobacterium tumefaciens.

7. the described application of wintersweet gene aspect the transgene tobacco drought resisting that comprises the thioesterase domain of claim 1.

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