CN105452281A

CN105452281A - Cotton trehalose synthetase tps-3, and coding gene and use thereof

Info

Publication number: CN105452281A
Application number: CN201380078632.3A
Authority: CN
Inventors: 陈文华; 孙超; 崔洪志
Original assignee: Genesis Seed Industry Co ltd
Current assignee: Genesis Seed Industry Co ltd
Priority date: 2013-09-17
Filing date: 2013-09-17
Publication date: 2016-03-30
Also published as: WO2015039261A1

Abstract

Disclosed are a trehalose synthetase TPS-3 derived from cotton and coding gene thereof, and use thereof in cultivating a transgenic plant having improved drought tolerance.

Description

Cotton trehalose synthetase tps-3, and coding gene and use thereof

Growing cotton, the present invention relates to vegetable protein and its encoding gene and application with applied technical field for trehalose synthetase TPS-3 and its encoding gene, more particularly to a kind of trehalose synthetase TPS-3 and its encoding gene from cotton, and its application in the genetically modified plants that drought tolerance is improved are cultivated.Background technology

The environment stresses such as temperature, salt marsh and arid can cause to seriously endanger to growing for higher plant, cause crop yield to reduce, quality decline is serious to threaten agricultural production and natural environment.Wherein influence of the arid to crop yield, first place is accounted in many natural adverse circumstances, and it is endangered equivalent to other disaster sums, be many areas be agricultural development bottleneck.According to statistics, world's arid, semiarid zone account for the 34% of land area;China's arid, semiarid zone account for the 52% of area, year area suffered from drought reach ten thousand hectares of 200-270, the national billion cubic meter of the annual water shortage in irrigation district about 30 receives hundred million kilograms of grain 350-400 because of water shortage less；Particularly China relationship and primary grain producing such as North China, northeast and northwest, is the area of China's water shortage most serious, and spring drought frequently reaches 10 years nine chances.

Drought resistance in plants belongs to the quantitative character of controlled by multiple genes mostly, is lacked using the drought resistance of conventional breeding methods Crop Improvement by cycle length, quality germplasm and is limited.The Preliminary Study of transcription group in recent years, protein science and gene expression regulation discloses the effect molecule mechanism of plant drouhgt stress.At present, the drought-resistant ability of plant is improved using drought stress related gene, the study hotspot and the important research direction of plant stress-resistance genetic engineering of plant stress-resistance molecular biology is had become.

Plant is by that can produce corresponding responsing reaction during environment stress, to reduce or eliminate the harm that environment stress comes to vegetational zone.This responsing reaction of plant is a complex process for being related to polygenes, multi signal approach and polygenes product.But for current research situation, because its mechanism is sufficiently complex, many plants still need to be studied to the biochemistry under adverse circumstance and physiological response mechanism.In the research in terms of the function and expression regulation of degeneration-resistant response gene important basis is provided by the research that network system is transmitted in the contact between the signaling pathways related to plant stress-resistance and whole signal.The content of the invention

The present inventor utilizes SSH (Subtractive hybridizations）With RACE (cDNA ends rapid amplifyings）The method being combined has cloned one and grown cotton the trehalose synthetase (encoding gene being named as herein, and determine its DNA sequence dna.And it was found that being conducted into after plant overexpression, the drought tolerance of transfer-gen plant is can obviously improve, and these characters can stablize heredity.

First aspect present invention provides a kind of trehalose synthetase GhTPS-3 of cotton encoding gene（It is named as herein GhTPS-3), its sequence is SEQ ID NO: 2.

Second aspect of the present invention provides a kind of recombinant expression carrier, and it contains the gene described in first aspect present invention and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector；Preferably, the carrier is the 2^-GhTPS-3-22 shown in accompanying drawing 2>QQ carriers.

Third aspect present invention provides a kind of recombinant cell, and it contains the recombinant expression carrier described in gene or second aspect of the present invention described in first aspect present invention；Preferably, the recombinant cell is restructuring agrobatcerium cell.

Fourth aspect present invention provides a kind of method of improvement drought resistance in plants, including：Recombinant expression carrier described in gene or second aspect of the present invention described in first aspect present invention is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is arabidopsis.

Fifth aspect present invention provides a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the gene or second aspect of the present invention described in first aspect present invention or plant tissue are cultivated under conditions of plant is effectively produced；Preferably, the plant is arabidopsis.

Gene, the recombinant expression carrier described in second aspect of the present invention or the recombinant cell described in third aspect present invention that sixth aspect present invention is provided described in first aspect present invention are used to improve drought resistance in plants and the purposes for plant breeding；Preferably, the plant is arabidopsis.

Seventh aspect present invention provides the protein of the gene code described in first aspect present invention, its amino acid sequence such as SEQ ID NO:Shown in 1.Description of drawings I is GhTPS-3 plant expression vector pSS-G/zr ^^^SOO) structure flow（a-b).

Fig. 2 is GhTPS-3 plant expression vector pSS-G/zr ^^^SOO) plasmid figure.

Fig. 3 GhTPS-3 T1 are for transgenic Arabidopsis plants（T1I5 the drought-enduring simulated experiment result of the non-transgenic Arabidopsis plant (CK)) and as control.（A is the normal growth Arabidopsis plant of 20 days；B is normal growth Osmotic treatment Arabidopsis plant of 14 days after 20 days）.

Fig. 4 is the protein expression the result of 1 generations of transgenosis T Arabidopsis plant and non-transgenic reference plant on transcriptional level.M is DNA Ladder Marker (DL2000, TakaRa), and 1-4 is the control of non-transgenic arabidopsis, and 5- 11 is drought-enduring 1 generations of transgenic arabidopsis T plant（It is followed successively by：The Ι 1 of Τ 1, the Ι 2 of Τ 1, the Ι 3 of Τ 1, the Ι 4 of Τ 1, the Ι 5 of Τ 1, the Ι 6 of Τ 1, the Ι 7 of Τ 1), 12- 16 is not drought-enduring transgenic arabidopsis Tl for plant. The present invention is further described with reference to non-limiting example for embodiment.The embodiment is not intended to limit the scope of the present invention merely for exemplary purpose.

The restriction enzyme in the unreceipted source mentioned in example below is purchased from New England Biolabs companies.In the present invention, if indicating and not having ambiguity within a context, ratio and percentage are calculated based on weight.

Cotton SSH library constructions under embodiment 1, drought stress.

Specific method is：

Subtracted library is built by Subtractive hybridization method using the method shown in the PCR-select cDNA Subtraction Kit of Clontech companies.Using the mRNA of the blade of the cotton seedling of Osmotic treatment as sample (Tester) in experimentation, control is used as using the mRNA of the blade of untreated cotton seedling（Driver).Specific steps are summarized as follows：

(1) material to be tested：

(National Cotton mid-term storehouse obtains unit Cotton research institute, Unified number to Ji cotton 14：ZM-30270) it is seeded on sterilized vermiculite, in 25 °C, 16 hours photoperiods illumination/8 hour dark（Light intensity 2000-3000 Lx) under the conditions of cultivate, 1/2MS fluid nutrient mediums are poured weekly（Containing 9.39 mM KN0₃, 0.625 mM KH₂P0₄, 10.3 mM NH₄NO₃ , 0.75 mM MgS0₄, 1.5 mM CaCl₂, 50 μ Μ KI, 100 μ Μ Η₃Β0₃, 100 μ Μ MnS0₄, 30 M ZnSO₄, 1 μ Μ Ν α₂Μο0₄, 0.1 μΜ CoCl₂, 100 μΜ Na₂EDTA, 100 M FeSO₄)-secondary.It is used to test when seedling strain is cultivated 1 month or so.

(2) material process：

Examination seedling is supplied to be divided into 2 groups, every group of 4 basins, per 1 plant of basin by above-mentioned.First group is control group, is cultivated under 25 °C, 16 hours photoperiods illumination/8 hour dark condition, normal to pour.Second group is Osmotic treatment group, 25 °C, cultivate under 16 hours photoperiods illumination/8 hour dark condition, stops pouring, processing 10 days, the blade of two groups of seedling apicals 1/3 of timely clip after being disposed, after liquid nitrogen quick freeze, is preserved in -70 °C of refrigerators.

(3) Total RNAs extraction：

Control group and each 0.1 g of the cotton leaf of Osmotic treatment group are taken respectively, use plant RNA extraction kit（It is purchased from

Invitrogen the total serum IgE of cotton leaf) is extracted.Gained total serum IgE is determined in 260 nm and 280 nm absorbance, OD with the ultraviolet specrophotometer U-2001 of HITACHI companies₂₆₀/OD₂₈₀Ratio is 1.8-2.0, shows that total serum IgE purity is higher；The integrality of total serum IgE is detected with 1.0% agarose gel electrophoresis, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.MRNA is separated using the Oligotex mRNA purification kits (purification of polyA+ RNA from total RNA) of Qiagen companies.

(4) Subtractive hybridization： By the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kits carries out Subtractive hybridization.Driver mRNA and Tester mRNA are first distinguished into reverse transcription, double-strand cDNA, then the progress subtractive hybridization using 2 Tester cDNA and 2 μ g Driver cDNA as parent material is obtained.Then the Tester cDNA after digestion are divided into joints different in two equal portions, connection by Tester cDNA and Driver cDNA Rsa I digestions 1.5 hours respectively under 37 °C of water-baths, and Driver cDNA are not connected to head.Two kinds of Tester cDNA for being connected with different joints are mixed with excessive Driver cDNA respectively, carry out positive subtractive hybridization for the first time.The product of two kinds of first time subtractives hybridization is mixed, then second of positive subtractive is carried out with the Driver cDNA that are newly denatured and is hybridized, the fragment of differential expression is then expanded by inhibition PCR twice, it is enriched with.

In order to have increased access to EST（Expressed sequence tag, EST) the validity of (unigene), avoid gene without restriction enzyme site and obtained sequence in non-translational region, digestion is carried out to Tester cDNA and Driver cDNA by above-mentioned steps with restriction endonuclease Haelll simultaneously for this experiment and priority carries out positive subtractive hybridization and inhibition PCR amplifications twice twice, finally merges second of inhibition PCR primer that two groups of positive subtractives hybridize cDNA fragments.

(5) structure of cDNA subtracted libraries and preliminary screening, clone, identification

According to pGEM-T Easy kits（Purchased from Promega) product description shown in method, the positive subtractive of above-mentioned merging is hybridized to second of PCR primer of cDNA fragments（Purified using QIAquick PCR Purification Kit, purchased from Qiagen) it is connected with pGEM-T Easy carriers, it is comprised the following steps that：Following ingredients are sequentially added into 200 μ PCR pipes：Positive subtractive after the merging of purifying hybridizes second of inhibition PCR primer, 31,2 χ Τ of μ, 4 DNA ligase buffer solutions 5 μ 1, the μ 1 of pGEM-T Easy carriers 1, the μ 1 of T4 DNA ligases 1 of cDNA fragments, is stayed overnight in 4 °C of connections.10 coupled reaction products are taken, is added in 100 competence e. coli jm109s (be purchased from TAKARA) and mixes, ice bath 30 minutes, 42 °C of heat shocks 60 seconds, ice bath 2 minutes, separately plus 250 μ L LB nutrient solutions（OXOID is purchased from containing 1% Tryptone, 0.5% Yeast Extract are purchased from OXOID, and 1% NaCl is purchased from traditional Chinese medicines）After be placed in 37 °C of shaking tables, with 225 revs/min of shaken cultivations 30 minutes, gained bacterium solution was subtracted library bacterium solution.Glycerol adding is saved backup to final concentration 20% (v/v) in -80 °C.

Subtracted library bacterium solution described in 200 is taken to be coated on containing 50 g/mL ampicillins（Purchased from Beijing Baeyer enlightening）, 40 ^glmL X-gal (the chloro- 3- indoles-β-D- galactosides of 5- bromo- 4）, 24 g/mL IPTG (isopropyl-β-D- Thiogalactopyranosides）（X-gal and IPTG are purchased from TAKARA) LB (ibid）Solid culture flat board（1.5% fine jade month purport, similarly hereinafter）On, 37 °C are cultivated 18 hours.Count diameter in culture plate>L mm clear white and blue colonies number, random 198 white colonies of picking (numbering：Gh-B001 to Gh-B198).All white colonies are inoculated in 96 porocyte culture plates of the LB fluid nutrient mediums containing 50 g/mL ampicillins respectively（CORNING in), glycerol adding is saved backup to final concentration 20% in -80 °C after 37 °C of overnight incubations.With nest-type PRC primer Primer 1 and Primer 2R, (the PCR-select cDNA Subtraction Kit kits of Clontech companies are carried）Bacterium solution PCR amplification checkings are carried out, 190 are obtained Then all positive colonies are being sent Invitrogen by individual positive colony（Shanghai）Trade Co., Ltd is sequenced.

(6) the cDNA sequencing analysis of differential cloning：

After the cDNA that DNA sequencing result is removed to carrier and indefinite sequence and redundancy, 135 EST are obtained, and (Unigene has 21 contigs through analysis, there is 114 single sequences.Find that wherein 48 EST (Unigene) have homologous sequence in GenBank through BlastN, 32 EST Unknown Functions are hypothesis albumen, separately there are 34 not obtain homologous matching, thus it is speculated that to be probably the shorter sequence in 3' or 5' ends non-translational region.The clone of embodiment 2, cotton trehalose synthetase encoding gene GhTPS-3.

Clone Gh-B77 removes after redundant DNA, and sequence is SEQ ID No:3, sequence analysis shows that the protein of the coding of the sequence belongs to trehalose synthetase, the corresponding total length encoding genes of clone GH-B77 is named as into GhTPS-3 herein, its corresponding albumen is named as 7 ^.

SEQ ID No: 3：

1 TCGAATAATC GATTGCTCAT ATTGGGATTT AATGCAACTT TAACTGAACG AGTGGATACC

61 CTTGGAAGGA AGGACAGTCA AATTAATGAA TGGGAGCCCA AGTTACGTCC AGATTTACAA

121 GAACCTTTGA GAAAACTTTG TGATGATCCG AAGACAACAG TTCTTATCCT CAGTGGAAGC

181 GCTAGAAATG TCCTGGATGA TAACTTCAGT GATTACAACT TGTCGTTGGC AGCCGAAAAT

241 GGGATGTTTT TACGAGTTAC AACGGGAGAT TGGATGACGA CAATGCCTGA AAACTTAAGT

301 ATGGAATGGG TGGATAGTGT TAAGAATGTA TTTGAATATT TTACCGATAG AACTCCTCGA

361 TCTCAGTTTG AGGTCCGAGA GACTTCACTC ATATGGAACT ATAAATATTC AGATGTTGAG

421 TTTGGAAGAC TTCAAGCGAG AGATCTGTTG CAGCATCTTT GGACTGGTCC GATCTCTAAC

481 GCATCTCTCG ATGTTGTCCA AGGTAGACGG TCTGTTGAGG GTGCCGCAAT CGATCGAATC

541 CTCGGAGAAA TAGTTCATAA CAAAGGCATG AAAGAACCTA TTGATTACGT CCTATGTATC

601 GGTCACTTTC TCACTAAGGA TGAAGACATT TATACATTTT TCGAGCCAGA GCTTCCCTCA

661 GAAGTCCCGG CTTCGGTAAG ACCCCAAGTA CCAACACAAG TTAGGACATC CACCGCTGGG

721 TCGAGGGCAG CTCTCCTTCT GAAACGAAAT TCTATGTCGG CTGTAGAACA GAACAAGAGC

781 TATTCCATAG ATGGAGATAC GTTGCACTTG ATGGTGACCG ATCGAATATC GTTTCAAGAG

841 GGTTCTTCAG TGCTTGATCT TCAGGCCAAC GATTAGTTTT CTTGCTCCGT TGCAAGGAAA

901 AGATCAAACT CGCGCTACCG CCTTCAATCA TCTGATGAAG TTGTGACACT CTTGAGAGAA

961 CTAGCAGATC ATCGTTGA

The clone of TPS-3 total length encoding genes

According to the SEQ ID No obtained:3 sequence analyses: SEQ ID No:3 be encoding gene TPS-3 3 terminal sequences.According to the SEQ ID NO obtained:3 sequences, design following three specific primers, are used as reverse transcription primer and 5 ' RACE specific primer.

GH-B77GSP1： SEQ ID NO: 4：

ACACTATCCACCCATTCCAT

GH-B77GSP1 : SEQ ID NO: 5： TCAGGCATTGTCGTCATCCA

GH-B77GSP3： SEQ ID NO:6:

TCGAATAATCGATTGCTCAT

Kit carries universal primer：

AAP: SEQ ID NO: 7：

GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP: SEQ ID NO: 8：

GGCCACGCGTCGACTAGTAC experimental procedures are operated by kit specification（5 ' RACE System for Rapid Amplification of cDNA Ends kits are purchased from Invitrogen companies）.

With GH-B77GSP1 (SEQ ID NO:4) it is reverse transcription primer, reverse transcription is carried out by template of cotton mRNA, cDNA templates is obtained, then adds Poly C tails according to the step in above-mentioned 5'RACE kit specifications, the amplification of first round PCR is carried out by template of the product after tailing, the primer is SEQ ID NO:4 and universal primer SEQ ID NO:7 (kit is carried, and I is a, c, g or t) that Hypoxanthine is modified, and is comprised the following steps that：

50 μ PCR reaction systems：5 μ Ι Ο Ε χ Buffer, the 3 μ 2.5 mM μ Ex of dNTP, the cDNA of 2.0 μm of RNA reverse transcriptions, 1.0 Taq (being purchased from TAKARA), 10 μ Μ primer SEQ ID NO:4 and SEQ ID NO:7 each 2.0 μ 1, and 35 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 45 seconds, and 55 °C are annealed 45 seconds, and 72 °C extend 90 seconds）, 72 °C extend 10 minutes.

The PCR primer of gained takes 2.0 μ as template after diluting 50 times with distilled water, with SEQ ID NO:5 and universal primer SEQ ID NO:8, which carry out second, takes turns PCR amplifications, comprises the following steps that：

50 y l PCR reaction systems：The first round PCR primer of 5 μ 1 10 X Ex Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ dilution, 1.0 w l Ex Taq, 10 μM of primer SEQ ID NO:5 and SEQ ID NO:8 each 2.0 μ 1, and 35 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 45 seconds, and 55 °C are annealed 45 seconds, and 72 °C extend 90 seconds））, 72 °C extend 10 minutes.Reclaim in second of PCR primer be about 1.4 Kbp sizes band（Gel Extraction Kit are purchased from OMEGA), and pGEM-T Easy Vector are connected to, being then transformed into JM109, (specific method is ibid）, random 6 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/mL ampicillins and cultivate respectively, and glycerol adding is to final concentration 20% (v/v) after 37 °C of overnight incubations, and -80 °C save backup.With primer SEQ ID NO:5 and 3' ends primer SEQ ID NO:6 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 6 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd is sequenced, and obtains the cDNA of the gene one section of 5' terminal sequence.

It is SEQ ID NO that the sequencing of 5'RACE product clonings 04 of gained, which obtains sequence,: 9： 1 GGGGGGGGGG TTTGCTGATG TCGTTAATGA ACATTATGAG GAAGGGGATG TTGTTTGGTG

61 CCATGATTAC CACCTAATGT TCCTTCCAAA ATGTCTAAAA GAACGGAACA GTAAAATGAA

121 AGTCGGTTGG TTCCTTCATA CTCCATTTCC TTCTTCGGAA ATCCATAGGA TGCTGCCATC

181 TCGGTTGGAG CTGCTGAGGT CCGTTCTTGC TGCTGATTTA GTAGGGCATT TTGTTAGTGC

241 TTGTACTCGT ATTCTCGGGC TGGAGGGCAC ACCTGAAGGA GTAGAGGATC AAGGAAAGCT

301 GACTCGCATT GCTGCATTTC CAGTAGGGAT TGATTCTGAA CGATTCATTA AAGCTCTTGA

361 ACTCCCTGAA GTACAAGATC ACATGAAGGT ACTGAAACAG AGGTTTGCAG GTCGAAAGGT

421 AATGTTAGGC GTTGATCGGC TTGATATGAT CAAGGGAATA CCCCAGAAGA TTTTGGCATT

481 TGAAAAATTC CTCCAGGAAA ATCCCAATTG GCGTAATAAG GTGGTTCTCC TTCAAATTGC

541 TGTCCCTACC AGGACCGATG TTCCTGAGTA TCAAAAGCTT ACAAGCCAGG TTCATGAGAT

601 TGTGGGGCGC ATTAATGGAA GATTTGGAAG CCTTACTGCT GTACCAATAC ATCATCTGGA

661 TCGTTCTCTT GATTTTCATG AATTATGTGC ACTGTACGCT GTTACAGATG TACCTCTTGT

721 GACATCTTTG AGGGATGGAA TGAACCTCGT CAGCTATGAG TACGTTGCCT GCCAAGAGTC

781 CAAGAAAGGG GTCCTTATAC TAAGTGAGTT TGCTGGGGCT GCTCAGTCTC TTGGTGCCGG

841 AGCCATTTTG GTTAATCCAT GGAATGTCAC TGAAGTTGCT TCTTCCATTG GTTATGCTTT

901 GAATATGCCA GCTGATGAAA GAGAAAAACG ACACCACCAT AACTTCAAGC ATGTGATTTC

961 ACATACAGCT CAAGAATGGG CTGCAACTTT TGTAAGTGAA CTTAATGTTA CCATTGTTGA

1021 AGCTCAACTG AGGACAAGAC AAATTCCCCC ACCACTCCCA ATTGAAGTTG CGGTTGATCG

1081 CTATTCATGT TCGAATAATC GATTGCTCAT ATTGGGATTT AATGCAACTT TAACTGAACG

1141 AGTGGATACC CTTGGAAGGA AGGACAGTCA AATTAATGAA TGGGAGCCCA AGTTACGTCC

1201 AGATTTACAA GAACCTTTGA GAAAACTTTG TGATGATCCG AAGACAACAG TTCTTATCCT

1261 CAGTGGAAGC GCTAGAAATG TCCTGGATGA TAACTTCAGT GATTACAACT TGTCGTTGGC

1321 AGCCGAAAAT GGGATGTTTT TACGAGTTAC AACGGGAGAT TGGATGACGA CAATGCCTGA are by 5'1.The ^^0 of 8 ￡ of sequence 0 10 that ￡ is obtained_:9, the sequence SEQ ID NO with acquisition:3 splicings, obtain SEQ ID NO: 10:

1 GGGGGGGGGG TTTGCTGATG TCGTTAATGA ACATTATGAG GAAGGGGATG TTGTTTGGTG

61 CCATGATTAC CACCTAATGT TCCTTCCAAA ATGTCTAAAA GAACGGAACA GTAAAATGAA

121 AGTCGGTTGG TTCCTTCATA CTCCATTTCC TTCTTCGGAA ATCCATAGGA TGCTGCCATC

181 TCGGTTGGAG CTGCTGAGGT CCGTTCTTGC TGCTGATTTA GTAGGGCATT TTGTTAGTGC

241 TTGTACTCGT ATTCTCGGGC TGGAGGGCAC ACCTGAAGGA GTAGAGGATC AAGGAAAGCT

301 GACTCGCATT GCTGCATTTC CAGTAGGGAT TGATTCTGAA CGATTCATTA AAGCTCTTGA

361 ACTCCCTGAA GTACAAGATC ACATGAAGGT ACTGAAACAG AGGTTTGCAG GTCGAAAGGT

421 AATGTTAGGC GTTGATCGGC TTGATATGAT CAAGGGAATA CCCCAGAAGA TTTTGGCATT

481 TGAAAAATTC CTCCAGGAAA ATCCCAATTG GCGTAATAAG GTGGTTCTCC TTCAAATTGC

541 TGTCCCTACC AGGACCGATG TTCCTGAGTA TCAAAAGCTT ACAAGCCAGG TTCATGAGAT

601 TGTGGGGCGC ATTAATGGAA GATTTGGAAG CCTTACTGCT GTACCAATAC ATCATCTGGA

661 TCGTTCTCTT GATTTTCATG AATTATGTGC ACTGTACGCT GTTACAGATG TACCTCTTGT

721 GACATCTTTG AGGGATGGAA TGAACCTCGT CAGCTATGAG TACGTTGCCT GCCAAGAGTC

781 CAAGAAAGGG GTCCTTATAC TAAGTGAGTT TGCTGGGGCT GCTCAGTCTC TTGGTGCCGG

841 AGCCATTTTG GTTAATCCAT GGAATGTCAC TGAAGTTGCT TCTTCCATTG GTTATGCTTT

901 GAATATGCCA GCTGATGAAA GAGAAAAACG ACACCACCAT AACTTCAAGC ATGTGATTTC

961 ACATACAGCT CAAGAATGGG CTGCAACTTT TGTAAGTGAA CTTAATGTTA CCATTGTTGA

1021 AGCTCAACTG AGGACAAGAC AAATTCCCCC ACCACTCCCA ATTGAAGTTG CGGTTGATCG

1081 CTATTCATGT TCGAATAATC GATTGCTCAT ATTGGGATTT AATGCAACTT TAACTGAACG

1141 AGTGGATACC CTTGGAAGGA AGGACAGTCA AATTAATGAA TGGGAGCCCA AGTTACGTCC

1201 AGATTTACAA GAACCTTTGA GAAAACTTTG TGATGATCCG AAGACAACAG TTCTTATCCT

1261 CAGTGGAAGC GCTAGAAATG TCCTGGATGA TAACTTCAGT GATTACAACT TGTCGTTGGC

1321 AGCCGAAAAT GGGATGTTTT TACGAGTTAC AACGGGAGAT TGGATGACGA CAATGCCTGA

1381 AAACTTAAGT ATGGAATGGG TGGATAGTGT TAAGAATGTA TTTGAATATT TTACCGATAG

1441 AACTCCTCGA TCTCAGTTTG AGGTCCGAGA GACTTCACTC ATATGGAACT ATAAATATTC 1501 AGATGTTGAG TTTGGAAGAC TTCAAGCGAG AGATCTGTTG CAGCATCTTT GGACTGGTCC

1561 GATCTCTAAC GCATCTCTCG ATGTTGTCCA AGGTAGACGG TCTGTTGAGG GTGCCGCAAT

1621 CGATCGAATC CTCGGAGAAA TAGTTCATAA CAAAGGCATG AAAGAACCTA TTGATTACGT

1681 CCTATGTATC GGTCACTTTC TCACTAAGGA TGAAGACATT TATACATTTT TCGAGCCAGA 1741 GCTTCCCTCA GAAGTCCCGG CTTCGGTAAG ACCCCAAGTA CCAACACAAG TTAGGACATC

1801 CACCGCTGGG TCGAGGGCAG CTCTCCTTCT GAAACGAAAT TCTATGTCGG CTGTAGAACA

1861 GAACAAGAGC TATTCCATAG ATGGAGATAC GTTGCACTTG ATGGTGACCG ATCGAATATC

1921 GTTTCAAGAG GGTTCTTCAG TGCTTGATCT TCAGGCCAAC GATTAGTTTT CTTGCTCCGT

1981 TGCAAGGAAA AGATCAAACT CGCGCTACCG CCTTCAATCA TCTGATGAAG TTGTGACACT 2041 CTTGAGAGAA CTAGCAGATC ATCGTTGA

According to SEQ ID NO:10 sequence retrievals are analyzed, SEQ ID NO:10 be not GhTPS-3 full length sequence.5 ' RACE need to be further carried out, so as to obtain full length gene according to SEQ ID NO:10 sequences, design following three specific primers, are used as reverse transcription primer and 5 ' RACE specific primer.

GH-B7702GSP1 : SEQ ID NO: 1 1 :

GCCTAACATTACCTTTCGACC GH-B7702GSP2: SEQ ID NO: 12:

GGGAGTTCAAGAGCTTTAATG GH-B7702GSP3 : SEQ ID NO: 13：

TTGCTGATGTCGTTAATGAAC experimental procedures are operated by kit specification（5'RACE System for Rapid Amplification of cDNA Ends kits are purchased from Invitrogen companies）.

With GH-B7702GSP1 (SEQ ID NO:11) it is reverse transcription primer, reverse transcription is carried out by template of cotton mRNA, cDNA templates is obtained, then adds Poly C tails according to the step in above-mentioned 5'RACE kit specifications, the amplification of first round PCR is carried out by template of the product after tailing, the primer is SEQ ID NO:11 and universal primer SEQ ID NO:7 (kit is carried, and I is a, c, g or t) that Hypoxanthine is modified, and is comprised the following steps that：

50 μ PCR reaction systems：5 μ Ι Ο Ε χ Buffer, the 3 μ 2.5 mM μ Ex of dNTP, the cDNA of 2.0 μm of RNA reverse transcriptions, 1.0 Taq (being purchased from TAKARA), 10 μ Μ primer SEQ ID NO:11 and SEQ ID NO:7 each 2.0 μ 1, and 35 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C of denaturation 45s, 55 °C are annealed 45 seconds, and 72 °C extend 90 seconds）, 72 °C extend 10 minutes.

The PCR primer of gained takes 2.0 μ as template after diluting 50 times with distilled water, with SEQ ID NO:12 and universal primer SEQ ID NO:8, which carry out second, takes turns PCR amplifications, comprises the following steps that：

50 y l PCR reaction systems：The first round PCR primer of 5 μ 1 10 X Ex Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ dilution, the Ex Taq of 1.0 μ 1,10 μM of primer SEQ ID NO:12 and SEQ ID NO:8 each 2.0 μ 1, and 35 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 45 seconds, and 55 °C are annealed 45 seconds, and 72 °C extend 90 seconds））, 72 °C extend 10 minutes.Reclaim second of PCR production It is about the band of 1.1 Kbp sizes in thing（Gel Extraction Kit are purchased from OMEGA), and it is connected to pGEM-T

Easy Vector, being then transformed into JM109, (specific method is ibid）, random 6 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/mL ampicillins and cultivate respectively, and glycerol adding is to final concentration 20% (v/v) after 37 °C of overnight incubations, and -80 °C save backup.With primer SEQ ID NO:12 and 3' ends primer SEQ ID NO:13 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 6 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd is sequenced, and obtains the cDNA of the gene one section of 5' terminal sequence.It is EQ ID NO that the sequencing of 5'RACE product clonings 4 of gained, which obtains sequence,: 14：

1 GGGGGGGGGG ATGTCAATTT CATCCCAAGA GATCTGCATT TTGCCTAACA AGAACAATCT

61 GCCTTTGTTG CAAACTAATG GTATGCCAGG GAACTTATAT AATATCTGTA GTAATCTCCC

121 TGCTACACCA ACAAGATTGG AAAGGCTTCT TAGAGACAGG GAGTTGAGGA AGTACTCCAA

181 GTGTTTGAAT GATTGTGAAG CTGAGTTACC ATCATCAACC CAAGATTGTT TTTCACCTGA

241 TCTTGAGACT TCAACTTGTT GGTTCAACGA TGAAGAACTT TTAGAACCTG TTTCAGCATC

301 AAGGTCTTTG AATGATGATG GCGAAAGACT CGATTTTCGA GCTTCAAAAC AACGGCTTTT

361 GGTGGTTGCT AATAGGTTGC CAGTCTCTGC AGTTAGGCAT GGGGAAGATT CTTGGCAACT

421 TGAGATGAGT GTTGGTGGCT TAGTCACTGC ACTTCTTGGT GTGAAGGAAT TTGATACCAG

481 ATGGATTGGT TGGGCTGGCG TGAATGTCCC TGATGAAATT GGACAAAAGG CATTAGCTAA

541 AGCTTTAGAT GAAAAGAGGT GCATACCGGT ATTTCTTGAT GAAGAAGATG TTCATCAATA

601 TTATAATGGA TATTGTAACA ACATATTATG GCCACTGTTC CACTATATCG GACTTCCGCA

661 AGAAGACCGC CTTGCGACTA CTCGGAGCTT TCAATCTCAG TTTGATGCTT ATAAGAAAGC

721 GAATCAGTTG TTTGCTGATG TCGTTAATGA ACATTATGAG GAAGGGGATG TTGTTTGGTG

781 CCATGATTAC CACCTAATGT TCCTTCCAAA ATGTCTAAAA GAACGGAACA GTAAAATGAA

841 AGTCGGTTGG TTCCTTCATA CTCCATTTCC TTCTTCGGAA ATCCATAGGA TGCTGCCATC

901 TCGGTTGGAG CTGCTGAGGT CCGTTCTTGC TGCTGATTTA GTAGGGCATT TTGTTAGTGC

961 TTGTACTCGT ATTCTCGGGC TGGAGGGCAC ACCTGAAGGA GTAGAGGATC AAGGAAAGCT

1021 GACTCGCATT GCTGCATTTC CAGTAGGGAT TGATTCTGAA CGATTCATTA AAGCTCTTGA

The sequence SEQ ID NO that 1081 ACTCCC obtain the 2nd 5 ' RACE of wheel:14, with splicing sequence SEQ ID NO:10 splicings, obtain SEQ ID NO: 15

1 GGGGGGGGGG ATGTCAATTT CATCCCAAGA GATCTGCATT TTGCCTAACA AGAACAATCT

61 GCCTTTGTTG CAAACTAATG GTATGCCAGG GAACTTATAT AATATCTGTA GTAATCTCCC

121 TGCTACACCA ACAAGATTGG AAAGGCTTCT TAGAGACAGG GAGTTGAGGA AGTACTCCAA

181 GTGTTTGAAT GATTGTGAAG CTGAGTTACC ATCATCAACC CAAGATTGTT TTTCACCTGA

241 TCTTGAGACT TCAACTTGTT GGTTCAACGA TGAAGAACTT TTAGAACCTG TTTCAGCATC

301 AAGGTCTTTG AATGATGATG GCGAAAGACT CGATTTTCGA GCTTCAAAAC AACGGCTTTT

361 GGTGGTTGCT AATAGGTTGC CAGTCTCTGC AGTTAGGCAT GGGGAAGATT CTTGGCAACT

421 TGAGATGAGT GTTGGTGGCT TAGTCACTGC ACTTCTTGGT GTGAAGGAAT TTGATACCAG

481 ATGGATTGGT TGGGCTGGCG TGAATGTCCC TGATGAAATT GGACAAAAGG CATTAGCTAA

541 AGCTTTAGAT GAAAAGAGGT GCATACCGGT ATTTCTTGAT GAAGAAGATG TTCATCAATA

601 TTATAATGGA TATTGTAACA ACATATTATG GCCACTGTTC CACTATATCG GACTTCCGCA

661 AGAAGACCGC CTTGCGACTA CTCGGAGCTT TCAATCTCAG TTTGATGCTT ATAAGAAAGC

721 GAATCAGTTG TTTGCTGATG TCGTTAATGA ACATTATGAG GAAGGGGATG TTGTTTGGTG

781 CCATGATTAC CACCTAATGT TCCTTCCAAA ATGTCTAAAA GAACGGAACA GTAAAATGAA

841 AGTCGGTTGG TTCCTTCATA CTCCATTTCC TTCTTCGGAA ATCCATAGGA TGCTGCCATC 901 TCGGTTGGAG CTGCTGAGGT TGCTGATTTA GTAGGGCATT TTGTTAGTGC

961 TTGTACTCGT ATTCTCGGGC TGGAGGGCAC ACCTGAAGGA GTAGAGGATC AAGGAAAGCT

1021 GACTCGCATT GCTGCATTTC CAGTAGGGAT TGATTCTGAA CGATTCATTA AAGCTCTTGA

1081 ACTCCCTGAA GTACAAGATC ACATGAAGGT ACTGAAACAG AGGTTTGCAG GTCGAAAGGT

1141 AATGTTAGGC GTTGATCGGC TTGATATGAT CAAGGGAATA CCCCAGAAGA TTTTGGCATT

1201 TGAAAAATTC CTCCAGGAAA ATCCCAATTG GCGTAATAAG GTGGTTCTCC TTCAAATTGC

1261 TGTCCCTACC AGGACCGATG TTCCTGAGTA TCAAAAGCTT ACAAGCCAGG TTCATGAGAT

1321 TGTGGGGCGC ATTAATGGAA GATTTGGAAG CCTTACTGCT GTACCAATAC ATCATCTGGA

1381 TCGTTCTCTT GATTTTCATG AATTATGTGC ACTGTACGCT GTTACAGATG TACCTCTTGT

1441 GACATCTTTG AGGGATGGAA TGAACCTCGT CAGCTATGAG TACGTTGCCT GCCAAGAGTC

1501 CAAGAAAGGG GTCCTTATAC TAAGTGAGTT TGCTGGGGCT GCTCAGTCTC TTGGTGCCGG

1561 AGCCATTTTG GTTAATCCAT GGAATGTCAC TGAAGTTGCT TCTTCCATTG GTTATGCTTT

1621 GAATATGCCA GCTGATGAAA GAGAAAAACG ACACCACCAT AACTTCAAGC ATGTGATTTC

1681 ACATACAGCT CAAGAATGGG CTGCAACTTT TGTAAGTGAA CTTAATGTTA CCATTGTTGA

1741 AGCTCAACTG AGGACAAGAC AAATTCCCCC ACCACTCCCA ATTGAAGTTG CGGTTGATCG

1801 CTATTCATGT TCGAATAATC GATTGCTCAT ATTGGGATTT AATGCAACTT TAACTGAACG

1861 AGTGGATACC CTTGGAAGGA AGGACAGTCA AATTAATGAA TGGGAGCCCA AGTTACGTCC

1921 AGATTTACAA GAACCTTTGA GAAAACTTTG TGATGATCCG AAGACAACAG TTCTTATCCT

1981 CAGTGGAAGC GCTAGAAATG TCCTGGATGA TAACTTCAGT GATTACAACT TGTCGTTGGC

2041 AGCCGAAAAT GGGATGTTTT TACGAGTTAC AACGGGAGAT TGGATGACGA CAATGCCTGA

2101 AAACTTAAGT ATGGAATGGG TGGATAGTGT TAAGAATGTA TTTGAATATT TTACCGATAG

2161 AACTCCTCGA TCTCAGTTTG AGGTCCGAGA GACTTCACTC ATATGGAACT ATAAATATTC

2221 AGATGTTGAG TTTGGAAGAC TTCAAGCGAG AGATCTGTTG CAGCATCTTT GGACTGGTCC

2281 GATCTCTAAC GCATCTCTCG ATGTTGTCCA AGGTAGACGG TCTGTTGAGG GTGCCGCAAT

2341 CGATCGAATC CTCGGAGAAA TAGTTCATAA CAAAGGCATG AAAGAACCTA TTGATTACGT

2401 CCTATGTATC GGTCACTTTC TCACTAAGGA TGAAGACATT TATACATTTT TCGAGCCAGA

2461 GCTTCCCTCA GAAGTCCCGG CTTCGGTAAG ACCCCAAGTA CCAACACAAG TTAGGACATC

2521 CACCGCTGGG TCGAGGGCAG CTCTCCTTCT GAAACGAAAT TCTATGTCGG CTGTAGAACA

2581 GAACAAGAGC TATTCCATAG ATGGAGATAC GTTGCACTTG ATGGTGACCG ATCGAATATC

2641 GTTTCAAGAG GGTTCTTCAG TGCTTGATCT TCAGGCCAAC GATTAGTTTT CTTGCTCCGT

2701 TGCAAGGAAA AGATCAAACT CGCGCTACCG CCTTCAATCA TCTGATGAAG TTGTGACACT

2761 CTTGAGAGAA CTAGCAGATC ATCGTTGA are according to SEQ ID NO:15 sequence retrievals are analyzed, SEQ ID NO:15 be G/z7-3 full length sequence.According to SEQ ID NO:15 sequences Design pair of primers are as follows：

GhTPS-3F-. SEQ ID NO: 16：

ATGTCAATTTCATCCCAAGAG

GhTPSSR: SEQ ID NO: 17：

TCAACGATGATCTGCTAGTTC AP : SEQ ID NO: 18：

GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT passes through SEQ ID NO:16 and SEQ ID NO:17 clone GhTPS-3 complete encoding sequences.

Cotton RNA is extracted, with primer SEQ ID NO:18 be reverse transcription primer, obtains cotton cDNA.Using Stratagene PfuUltra II Fusion HS DNA Polymerase, performing PCR is entered using the cDNA of cotton as template Reaction.50 l PCR reaction systems：5 μ 1 10 X PfuUltra II reaction Buffer, 0.5 μ 1 25 mM dNTP,

10 μM of the PfuUltra II Fusion HS DNA Polymerase of 2.0 μ 1 cDNA, 1.0 μ 1 primer SEQ ID NO:16 and SEQ ID NO:17 each 2.0 μ 1, and 37.5 μ distilled water.PCR reaction conditions：95 °C of pre-degenerations 2 minutes, 35 circulations（95 °C are denatured 25 seconds, and 55 °C are annealed 25 seconds, and 72 °C extend 2 minutes）, 72 °C extend 5 minutes.

Pcr amplification product adds A tails：PCR primer moisturizing first takes out one time with chloroform and removes removing protein to 400 μ 1, draws supernatant and adds the μ 1 of 3 Μ sodium acetate solutions 40, plus 2 times of absolute ethyl alcohol, -20 °C are placed 10 minutes, centrifugation, supernatant is removed, is dried, is dissolved with 21 μ distilled waters.Add 2.5 μ 1 10 X Ex Buffer, 0.5 μ 15 mM dATP, 1.0 μ Ex Taq.Reaction condition：70 °C are reacted 30 minutes.The DNA fragmentation for obtaining about 2.8 Kbp is reclaimed（Omega QIAquick Gel Extraction Kits）, it is connected on pGEM T-easy carriers（Obtain G/z7-3-pGEM plasmids）, JM109 is then converted, random 6 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/mL ampicillins and cultivated respectively, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup.With primer SEQ ID NO:16 and SEQ ID NO:17 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 6 positive colonies are obtained, Invitrogen is delivered to（Shanghai）Trade Co., Ltd is sequenced, and sequence is SEQ ID NO:2, the amino acid sequence of its albumen encoded is SEQ ID NO: 1

The amino acid sequence of TPS-3 albumen： SEQ ID NO: l

1 MS ISSQEICI LPNKNNLALL QTNGMPGNLY NICSNLPATP TRLERLLRDR ELRKYSKCLN

61 DCEAELPSST QDCFSPDLET STCWFNDEEL LEPVSASRSL NDDGERLDFR ASKQRLLWA 121 NRLPVSAVRH GEDSWQLEMS VGGLVTALLG VKEFDTRWIG WAGVNVPDEI GQKALAKALD

181 EKRCI PVFLD EEWHQYYNG YCNNILWPLF HYIGLPQEDR LATTRSFQSQ FDAYKKANQL

241 FADWNEHYE EGDWWCHDY HLMFLPKCLK ERNSK KVGW FLHTPFPSSE IHRMLPSRLE

301 LLRSVLAADL VGHFVSACTR ILGLEGTPEG VEDQGKLTRI AAFPVGIDSE RFIKALELPE

361 VQDHMKVLKQ RFAGRKVMLG VDRLDMIKGI PQKILAFEKF LEENPNWRNK WLLQIAVPT 421 RTDVPEYQKL TSQVHEIVGR INGRFGSLTA VPIHHLDRSL DFHELCALYA VTDVALVTSL

The 481 beautiful P of RDGMNLVSYE YVACQESKKG VLILSEFAGA AQSLGAGAIL VNPWNVTEVA SS IGYAL

541 ADEREKRHHH NFKHVISHTA QEWAATFVSE LNVTIVEAQL RTRQI PPPLP IEVAVDRYSC

601 SNNRLLILGF NATLTEPVDT LGRKDSQINE WEPKLRPDLQ EPLRKLCDDP KTTVLILSGS

661 ARNVLDDNFS DYNLWLAAEN GMFLRVTTGD WMTTMPENLS MEWVDSVKNV FEYFTDRTPR 721 SQFEVRETSL IWNYKYSDVE FGRLQARDLL QHLWTGPISN ASLDWQGRR SVEGAAIDRI

781 LGEIVHNKGM KEPIDYVLCI GHFLTKDEDI YTFFEPELPS EVPASVRPQV PTQVRTSTAG

841 SRAALLLKRN SMSAVEQNKS YS IDGDTLHL MVTDRISFQE GSSVLDLQAN DYFSCSVARK

The nucleotide sequence of 901 RSNSRYRLQS SDEWTLLRE LADHR encoding genes： SEQ ID NO: 2

ATGTCAATTT CATCCCAAGA GATCTGCATT TTGCCTAACA AGAACAATCT GGCTTTGTTG

61 CAAACTAATG GTATGCCAGG GAACTTATAT AATATCTGTA GTAATCTCCC TGCTACACCA 121 ACAAGATTGG AAAGGCTTCT TAGAGACAGG GAGTTGAGGA AGTACTCCAA GTGTTTGAAT 181 GATTGTGAAG CTGAGTTACC ATCATCAACC CAAGATTGTT TTTCACCTGA TCTTGAGACT 241 TCAACTTGTT GGTTCAACGA TGAAGAACTT TTAGAACCTG TTTCAGCATC AAGGTCTTTG 301 AATGATGATG GCGAAAGACT CGATTTTCGA GCTTCAAAAC AACGGCTTTT GGTGGTTGCT 361, AATAGGTTGC, CAGTCTCTGC, AGTTAGGCAT, GGGGAAGATT, CTTGGCAACT, TGAGATGAGT, 421, GTTGGTGGCT, TAGTCACTGC, ACTTCTTGGT, GTGAAGGAAT, TTGATACCAG, ATGGATTGGT, 481, TGGGCTGGCG, TGAATGTCCC, TGATGAAATT, GGACAAAAGG, CATTAGCTAA, AGCTTTAGAT, 541, GAAAAGAGGT, GCATACCGGT, ATTTCTTGAT, GAAGAAGTTG, TTCATCAATA, TTATAATGGA, 601, TATTGTAACA, ACATATTATG, GCCACTGTTC, CACTATATCG, GACTTCCGCA, AGAAGACCGC, 661, CTTGCGACTA, CTCGGAGCTT, TCAATCTCAG, TTTGATGCTT, ATAAGAAAGC, GAATCAGTTG, 721, TTTGCTGATG, TCGTTAATGA, ACATTATGAG, GAAGGGGATG, TTGTTTGGTG, CCATGATTAC, 781, CACCTAATGT, TCCTTCCAAA, ATGTCTAAAA, GAACGGAACA, GTAAAATGAA, AGTCGGTTGG, 841, TTCCTTCATA, CTCCATTTCC, TTCTTCGGAA, ATCCATAGGA, TGCTGCCATC, TCGGTTGGAG, 901, CTGCTGAGGT, CCGTTCTTGC, TGCTGATTTA, GTAGGGCATT, TTGTTAGTGC, TTGTACTCGT, 961, ATTCTCGGGC, TGGAGGGCAC, ACCTGAAGGA, GTAGAGGATC, AAGGAAAGCT, GACTCGCATT, 1021, GCTGCATTTC, CAGTAGGGAT, TGATTCTGAA, CGATTCATTA, AAGCTCTTGA, ACTCCCTGAA, 1081, GTACAAGATC, ACATGAAGGT, ACTGAAACAG, AGGTTTGCAG, GTCGAAAGGT, AATGTTAGGC, 1141, GTTGATCGGC, TTGATATGAT, CAAGGGAATA, CCCCAGAAGA, TTTTGGCATT, TGAAAAATTC, 1201, CTCGAGGAAA, ATCCCAATTG, GCGTAATAAG, GTGGTTCTCC, TTCAAATTGC, TGTCCCTACC, 1261, AGGACCGATG, TTCCTGAGTA, TCAAAAGCTT, ACAAGCCAGG, TTCATGAGAT, TGTGGGGCGC, 1321, ATTAATGGAA, GATTTGGAAG, CCTTACTGCT, GTACCAATAC, ATCATCTGGA, TCGTTCTCTT, 1381, GATTTTCATG, AATTATGTGC, ACTGTACGCT, GTTACAGATG, TAGCTCTTGT, GACATCTTTG, 1441, AGGGATGGAA, TGAACCTCGT, CAGCTATGAG, TACGTTGCCT, GCCAAGAGTC, CAAGAAAGGG, 1501, GTCCTTATAC, TAAGTGAGTT, TGCTGGGGCT, GCTCAGTCTC, TTGGTGCCGG, AGCCATTTTG, 1561, GTTAATCCAT, GGAATGTCAC, TGAAGTTGCT, TCTTCCATTG, GTTATGCTTT, GAATATGCCA, 1621, GCTGATGAAA, GAGAAAAACG, ACACCACCAT, AACTTCAAGC, ATGTGATTTC, ACATACAGCT, 1681, CAAGAATGGG, CTGCAACTTT, TGTAAGTGAA, CTTAATGTTA, CCATTGTTGA, AGCTCAACTG, 1741, AGGACAAGAC, AAATTCCCCC, ACCACTCCCA, ATTGAAGTTG, CGGTTGATCG, CTATTCATGT, 1801, TCGAATAATC, GATTGCTCAT, ATTGGGATTT, AATGCAACTT, TAACTGAACC, AGTGGATACC, 1861, CTTGGAAGGA, AGGACAGTCA, AATTAATGAA, TGGGAGCCCA, AGTTACGTCC, AGATTTACAA, 1921, GAACCTTTGA, GAAAACTTTG, TGATGATCCG, AAGACAACAG, TTCTTATCCT, CAGTGGAAGC, 1981, GCTAGAAATG, TCCTGGATGA, TAACTTCAGT, GATTACAACT, TGTGGTTGGC, AGCCGAAAAT, 2041, GGGATGTTTT, TACGAGTTAC, AACGGGAGAT, TGGATGACGA, CAATGCCTGA, AAACTTAAGT, 2101, ATGGAATGGG, TGGATAGTGT, TAAGAATGTA, TTTGAATATT, TTACCGATAG, AACTCCTCGA, 2161, TCTCAGTTTG, AGGTCCGAGA, GACTTCACTC, ATATGGAACT, ATAAATATTC, AGATGTTGAG, 2221, TTTGGAAGAC, TTCAAGCGAG, AGATCTGTTG, CAGCATCTTT, GGACTGGTCC, GATCTCTAAC, 2281, GCATCTCTCG, ATGTTGTCCA, AGGTAGACGG, TCTGTTGAGG, GTGCCGCAAT, CGATCGAATC, 2341, CTCGGAGAAA, TAGTTCATAA, CAAAGGCATG, AAAGAACCTA, TTGATTACGT, CCTATGTATC, 2401, GGTCACTTTC, TCACTAAGGA, TGAAGACATT, TATACATTTT, TCGAGCCAGA, GCTTCCCTCA, 2461, GAAGTCCCGG, CTTCGGTAAG, ACCCCAAGTA, CCAACACAAG, TTAGGACATC, CACCGCTGGG, 2521, TCGAGGGCAG, CTCTCCTTCT, GAAACGAAAT, TCTATGTCGG, CTGTAGAACA, GAACAAGAGC, 2581, TATTCCATAG, ATGGAGATAC, GTTGCACTTG, ATGGTGACCG, ATCGAATATC, GTTTCAAGAG, 2641, GGTTCTTCAG, TGCTTGATCT, TCAGGCCAAC, GATTACTTTT, CTTGCTCCGT, TGCAAGGAAA, 2701, AGATCAAACT, CGCGCTACCG, CCTTCAATCA, TCTGATGAAG, TTGTGACACT, CTTGAGAGAA, 2761, CTAGCAGATC, ATCGTTGA, embodiment, 3,GTirPS-j gene plant expression vector establishments.

Plant binary expression vector pCAMBIA2300 is selected (to be purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）As plant expression vector, the 35S promoter that Ρ Τ Π genes contain double enhancers is replaced with Pnos promoters, to reduce expression of the Ν Ρ Τ Π albumen in plant.Selection contains 35S promoter and terminator the Tnos promoter and terminator respectively as GhTPS-3 genes of double enhancers.

With primer SEQ ID NO:19 and SEQ ID NO:20 (are purchased from ocean Science and Technology Ltd. of Beijing China with plant expression vector pBI121）For template amplification Pnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases. 50 μΐ PCR reaction systems：10 μ 5 xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ pBI121,1.0 μ PrimeSTAR,

Ι Ο μ Μ primer SEQ ID NO:The ^^0 of 19 standing grain P, 8 ￡ 0 10:20 each 2.0 ^, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 56 °C are annealed 30 seconds, and 72 °C extend 30 seconds）, 72 °C extend 10 minutes.PCAMBIA2300 (Promega, T4 ligase box) is connected to as the PCR products by obtained by after EcoRI, Bglll digestion and obtains pCAMBIA2300-l.

SEQ ID NO: 19 ：

GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO:20 :

ATCCAGATCTAGATCCGGTGCAGATTATTTG SEQ ID NO:21 standing grain P SEQ ID NO:22 using pBI121 as template amplification Tnos, using TaKaRa's

PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 μ 5 xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ pBI121,1.0 μ PrimeSTAR 10 μ Μ primer SEQ ID NO:21 standing grain mouthful SEQ ID NO:22 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, (94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 30 seconds for 33 circulations）, 72 °C extend 10 minutes.PCAMBIA2300-l (Promega T4 ligase boxes are connected to as the PCR products by obtained by after Kpnl, EcoRI digestion）Obtain pCAMBIA2300-2.

SEQ ID NO: 21：

CGGGG7MCCGAATTTCCCCGATCGTTCAAA

SEQ ID NO: 22：

TCKGAA rrCCC AGTGAATTCCCGATCT AGT A

SEQ ID NO:23 and SEQ ID NO:24 using pCAMBIA2300 plasmids as template amplification arabidopsis 35S promoter.Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 μ 5 xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ dilute 50 times of pCAMBIA2300 plasmids, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:23 and SEQ ID NO:24 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, (94 °C are denatured 30 seconds, and 50 °C are annealed 30 seconds, and 72 °C extend 30 seconds for 33 circulations；), 72 °C extend 10 minutes.It is connected to as the PCR primer by obtained by after HindIII, Xbal digestion（Connection method is ibid）PCAMBIA2300-2 obtains pCAMBIA2300-3

SEQ ID NO: 23：

ACT^GCrJATGGTGGAGCACGACACTCT

SEQ ID NO: 24：

GCJC7¾G^AGAGATAGATTTGTAGAGAGAGAC SEQ ID NO:25 and SEQ ID NO:(template is the positive that embodiment 2 is obtained to 26 amplification GhTPS-3

G/z7-3-pGEM plasmids）, using Stratagene PfuUltra II Fusion HS DNA Polymerase.50 μ PCR reaction systems：5 μ lO PfuUltra II reaction Buffer, 0.5 μ 25 mM dNTP, 2.0 μ GhTPS-3-pGEM plasmids, the μ Μ of 1.0 μ PfuUltra II Fusion HS DNA Polymerase 10 primer SEQ ID NO:25 standing grain P SEQ ID NO:26 each 2.0 μ 1, and 37.5 μ distilled water.PCR reaction conditions：95 °C of pre-degenerations 2 minutes, (95 °C are denatured 25 seconds, and 53 °C are annealed 25 seconds, and 72 °C extend 2 minutes, and 72 °C extend 5 minutes for 35 circulations.Connected as the PCR primer by obtained by after XbaI, KpnI digestion（Connection method is ibid）To pCAMBIA2300-3, plant expression vector 35S-GhTPS-3-2300 is obtained.

SEQ ID NO: 25：

AArC7¾&4ATGCCTGGAAACAAGTACAAC

SEQ ID NO: 26：

GCGG¾CCTCAAGTGCCTTTTGCTAGTC

Embodiment 4,358- (^7 ^-- 2300 expression vectors convert Agrobacterium.

The preparation of Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competent cell：Agrobacterium LBA4404 was drawn to single spot inoculation on the LB solid mediums containing 50 g/ml rifampins and 50 g/ml streptomysins in 1-2 days in advance, 28 °C are cultivated 1 to 2 day.Picking single bacterium colony is inoculated in 5 ml containing 50 μ_§The rifampins of/ι η 1 and 50 μ_§In the LB fluid nutrient mediums of the streptomysins of/ι η 1, overnight incubation is shaken under 28 °C（About 12-16 hours）To OD_6(K)It is worth for 0.4, formation seed bacterium solution.Take the bacterium solution after 5 ml activation（1 :20 ratio）In the LB fluid nutrient mediums for being inoculated in the same concentration antibiotic of 100 ml, 28 °C of shakes cultivate 2-2.5 hours to OD_6(K)=0.8.Ice bath bacterium solution 10 minutes, shook up once every 3 minutes, made bacterium even into resting state.Centrifuged 10 minutes in 4000 g under 4 °C, abandon supernatant；Add 4000 g under 10% (v/v) glycerine resuspension thalline of 1 ml ice precoolings, 4 °C to centrifuge 10 minutes, collect precipitation；Ice-cold 10% (v/v) glycerine repeats to wash 3-4 times；Adding 10% (v/v) glycerine of appropriate ice bath precooling, suspended bacterial is precipitated again, is dispensed, is saved backup in -70 °C with 40 μ/pipe.

Convert Agrobacterium：Melt competent cell on ice ,-the GhTPS-3-2 of the positive 2 of gained in 1 μ embodiments 3, plasmid, ice bath about 10 minutes after mixing are added into 40 μ competent cell.The mixture of the competent cell and DNA is transferred in the electric shock of ice precooling cup with liquid-transfering gun, rapping makes suspension reach bottom, has been careful not to bubble.To be shocked by electricity cup（Purchased from Bio-Rad) it is put on the slideway of electroporation chamber, promote slideway to put electric shock cup to electroporation chamber base electrode.Using the electric shock cup of 0.1 cm specifications, MicroPuMUer (being purchased from Bio-Rad) program is set to " Agr ", and electric shock is once.Electric shock cup is taken out immediately, adds the LB culture mediums of 28 °C of preheatings.It is quickly soft to be beaten cell with liquid-transfering gun.Suspension is transferred to 1.5 ml centrifuge tube, 28 °C, 225 rpm are cultivated 1 hour.100-200 μ bacterium solution is taken to be coated on corresponding resistance screening culture medium flat plate（LB solid mediums, containing 50 μ_§The rifampins of/ι η 1,50 g/ml chains Mycin, 50 μ_§The kanamycins of/ι η 1）, 28 °C of cultures.Positive transformants clone is screened, and its bacterium solution is saved backup in -70 °C.Embodiment 5, utilize Agrobacterium-medialed transformation method obtain transgenic arabidopsis.

Plant culture to be transformed：Arabidopsis seed（Colombia's type, the arabidopsis Biological Resource Center from Ohio State Univ-Columbus USA）Sowing is in peat soil, after 4 °C of low-temperature treatments 3 days, is placed in 23 °C, germinates in the dark incubator in 16 hours illumination/8 hour.It is transplanted to after 7-10 days equipped with peat soil and vermiculite（Volume ratio 3:1) bore is in 7.5 cm polypots, every 6 plants of pot culture kind is placed in 23 °C, the dark incubator in 16 hours illumination/8 hour and grown.The ml of nutrient solution 40 is poured before transplanting per alms bowl, backsight soil moisture is transplanted and keeps the skin wet in time.In the appropriate nutrient solution of growth period.It is every on demand₃-₄Zhou Yici（Or the time is longer）.In order to obtain more bud on each plant, first inflorescence is cut off after most of first inflorescence of plant are formed, apical dominance is released, promotes the synchronous appearance of multiple secondary inflorescences.When most of inflorescences about 1-10 cm are high（Cut off after first inflorescence 4-8 days）When prepare contaminate.

The culture of Agrobacterium：Take out after the bacterium liquid activation of Agrobacterium positive transformants clone of conservation in embodiment 4, picking Agrobacterium single bacterium colony is inoculated into the sterile LB fluid nutrient mediums of 10 mL（Containing 75 mg/ L rifampins, 100 mg/ L streptomysins and 100 mg/ L kanamycins）, the lower 250 revs/min of shakings incubated overnight of 28 °C of constant temperature.Resulting bacterium solution is inoculated into the 200 mL equally LB fluid nutrient mediums containing above-mentioned antibiotic by 1%-2% volume ratio again, 28 °C of constant temperature shakings make the concentration of Agrobacterium reach OD_6(K)=1.8, then centrifuged 15 minutes at 4 °C lower 3000 revs/min, with dip-dye culture medium after abandoning supernatant（The dip-dye culture medium contains 5.0% sucrose and 0.05% (500 μ Ι Τ) Silwet L-77) suspend Agrobacterium again, is suspended into OD_6QQAbout 0.80.

The dip-dye of inflorescence：The above-mentioned dip-dye culture medium containing Agrobacterium is added in big mouth container, the dip-dye culture medium containing Agrobacterium described in 200-300 mL is added in each cm of bore 9 container to be used to contaminate.Plant is inverted, aerial tissues is fully immersed in agrobacterium suspension 3-5 seconds, and to be gently agitated for.There should be one layer of liquid film after infiltration on plant.The plant contaminated is placed in vinyl disc, with clean plastics or preservative film covering with moisturizing, is then placed within dim light or dark place is stayed overnight, note carefully preventing direct sunlight plant.Remove covering within about 12-24 hours after processing.Normal culture plant, plant further growth 3-5 weeks, until silique browning is dried.Seed is harvested, and by seed centrifuge tube in 4 °C of lower dry storages.

Transgenic seed is screened：Prepare and contain 1/4 MS a great number of elements（4.695 mM KN0₃, 0.3125 mM KH₂P0₄, 5.15 mM H₄N0₃, 0.375mM MgS0₄, 0.75mM CaCl₂, 25 μ Μ Κ Ι, 50 μ Μ Η₃ΒΟ₃, 50 M MnS0₄, 15 M ZnS0₄, 0.5 μ Μ Ν α₂Μο0₄, 0.05 M CoCl₂, 50 μ Μ Na₂EDTA, 50 M FeSO₄) the aqueous solution, add 0.8 % agar powders, be heated to agar with micro-wave oven and dissolve completely, to be cooled to 50 °C or so, add the desired amount of final concentration of 50 rn U¹Kanamycins, per 25 mL are poured into culture dish after shaking up, putting can sow after experimental bench cooled and solidified.Load weighted seed is poured on a copy paper, use finger tap copy paper, seed is equably sowed on agaropectin, cover culture dish lid, after putting 4 °C of refrigerator cold treatments 72 hours, move to 23 °C, germinate in the dark incubator in 16 hours illumination/8 hour, resistance seedling is transplanted in Nutrition Soil by periodic statistical germination and growth of seedling situation in time. Backsight soil moisture is transplanted to keep the skin wet in time.In the appropriate nutrient solution of growth period.The g of Arabidopsis leaf 0.1 of growth 20 days is taken, DNA is extracted, with SEQ ID NO:16 and SEQ ID NO:17 amplification GhTPS-3:(50 μ PCR reaction systems：5 μ Ι Ο Ε χ Buffer, 3 μ 2.5 mM dNTP, 2.0 μ DNA, 1.0 μ Ex Taq, 10 μ Μ primer SEQ ID NO:16 and SEQ ID NO:17 each 2.0 μ 1, and 35 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 45 seconds, and 53 °C are annealed 45 seconds, and 72 °C extend 2 minutes）, 72 °C extend 7 minutes）, the PCR plant for being accredited as the positive are numbered（), T1I1-T1I12 and preserve.The drought-enduring simulated experiment and Function Identification of embodiment 6, overexpression GhTPS-3 transgenic arabidopsis T1 for plant.Sterilized vermiculite is impregnated with 1/2MS culture mediums.T1I1-T1I6 and control arabidopsis seed are sowed on vermiculite respectively, and 10 seeds, 25 °C, optical culture/14 hour light culture circulation in 10 hours are sowed per basin, pour a 1/2MS within every 7 days, after culture 20 days, per 4 more consistent seedlings of basin reservation size, for arid experiment.Transgenic arabidopsis, control arabidopsis arid 14 days（Do not water）, 25 °C, optical culture/14 hour light culture circulation in 10 hours.T1 is for transfer-gen plant（The plant that T0 grows up to for the seed of transfer-gen plant）Identification of Drought show that adjoining tree is all wilted seriously, and six strains of T1I1, T1I2, T1I3, T1I4, T1I5, T1I6 totally 28（Per each 4-5 of strain）25 can survive and continued growth shows obvious drought tolerance in arabidopsis（Referring to Fig. 3 a and 3b, by taking T1I5 as an example, the Ι 2 of T1I1, Τ 1, the Ι 3 of Τ 1, the Ι 4 of Τ 1, T1I6 result it is similar with T1I5, be not shown here）.Embodiment 7, the checking GhTPS-3 protein expressions on transcriptional level.

Control Arabidopsis plant, drought-enduring transgenic arabidopsis T1 are taken respectively for plant（It is belonging respectively to the Ι 2 of T1I1, Τ 1, the Ι 3 of Τ 1, the Ι 4 of Τ 1, the Ι 5 of Τ 1, six strains of T1I6）Each 0.05 g of the arid blade of 10 days of not drought-enduring 1 generations of transgenic arabidopsis Τ plant, uses plant RNA extraction kit（Invitrogen) the total serum IgE extracted.Total serum IgE is determined in 260 nm and 280 nm absorbance with the ultraviolet specrophotometer U-2001 of HITACHI companies, calculates each RNA concentration.Trying the reverse transcription of method progress shown in neat Ll boxes Superscript III Reverse Transcriptase according to Invitrogen reverse transcriptions, (2 μ g total serum IgEs are used as template, reverse transcription primer SEQ ID NO: 18).Pass through SEQ ID NO:16 and SEQ ID NO:17 amplification GhTPS-3, detect albumen relative expression's situation.

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of reverse transcription.The Ρ Ο reaction systems of 50 μ 1：10 μ 5 xPS Buffer, 3 μ 2.5 mM dNTP, 2.0 μ cDNA, 1.0 μ PrimeSTAR 10 μ Μ primer SEQ ID NO:16 and SEQ ID NO:17 each 2.0 μ 1, and 30 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 29 circulations（94 °C are denatured 45 seconds, and 53 °C are annealed 45 seconds, and 72 °C extend 2 minutes）, 72 °C extend 10 minutes.

Product electrophoresis result is as shown in Figure 4：M is DNA Ladder Marker (DL2000, TakaRa), and 1-4 is non-turn Gene arabidopsis is compareed, and 5-11 is drought-enduring transgenic arabidopsis T1 for plant（It is followed successively by： T1I1、 Τ1Ι2、 Τ1Ι3、

The Ι 4 of Τ 1, the Ι 5 of Τ 1, the Ι 6 of Τ 1, T1I7), 12-16 is not drought-enduring 1 generations of transgenic arabidopsis Τ plant.The size of PCR primer electrophoretic band shown in figure and GhTPS-3's is in the same size（About 2.8 Kbp).As a result show, control arabidopsis does not have GhTPS-3 transcriptions, and drought-enduring transgenic arabidopsis T1 is stronger for the transcription of GhTPS-3 in plant, not drought-enduring transgenic arabidopsis T1 is transcribed very weak or do not transcribed for plant.

Claims

Claims

1. a kind of albumen for the gene code for encoding cotton trehalose synthetase, its amino acid sequence is SEQ ID NO: l o

2. encode the gene of albumen described in claim 1, its nucleotide sequence such as SEQ ID NO:Shown in 2.

3. a kind of recombinant expression carrier, it contains the gene described in claim 2 and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector.

4. the recombinant expression carrier described in claim 3, it is the 35S-GM ^-3-2300 carriers shown in Fig. 2.

5. a kind of recombinant cell, it contains the recombinant expression carrier described in gene or claim 3 or 4 described in claim 2；Preferably, the recombinant cell is restructuring agrobatcerium cell.

6. a kind of method of improvement drought resistance in plants, including：Recombinant expression carrier described in gene or Claims 2 or 3 described in claim 1 is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is arabidopsis.

7. a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the gene or claim 3 or 4 described in claim 2 or plant tissue are cultivated under conditions of plant is effectively produced.

8. the method described in claim 7, wherein the plant is arabidopsis.

9. the recombinant expression carrier described in gene, claim 3 or 4 described in claim 2 or the recombinant cell described in claim 5 are used to improve drought resistance in plants or the purposes for plant breeding.

10. the purposes described in claim 9, wherein the plant is arabidopsis.