CN105051059A

CN105051059A - Cotton dehydrin protein, coding gene of same, and application thereof

Info

Publication number: CN105051059A
Application number: CN201380074552.0A
Authority: CN
Inventors: 陈文华; 孙超; 何云蔚; 崔洪志
Original assignee: Genesis Seed Industry Co ltd
Current assignee: Genesis Seed Industry Co ltd
Priority date: 2013-05-28
Filing date: 2013-05-28
Publication date: 2015-11-11
Anticipated expiration: 2033-05-28
Also published as: CN105051059B; WO2014190489A1

Abstract

A cotton-sourced dehydrin protein dehydrin09, a coding gene of same, and an application thereof in cultivating a transgenic plant of increased drought tolerance.

Description

Cotton dehydrin protein, coding gene of same, and application thereof

One grow cotton dehydrin protein and its encoding gene with applied technical field the present invention relates to vegetable protein and its encoding gene and application, more particularly to one derive from cotton dehydrin protein dehydri_n09 and its encoding gene, and its application in the genetically modified plants that drought tolerance is improved are cultivated.Technical background Dehydrins are the classes in LEA protein, are widely present in each histoorgan and the Embryos Development of Plant later stage of plant.Dehydrins are the class high-hydrophilic protected proteins that plant synthesizes when by abiotic stress such as low temperature, arid and high salts, and injured function is exempted from protection nucleic acid, intracellular protein and membrane structure.Many researchs are had confirmed under abiotic stress, are existed and are closely contacted between the expression of plant dehydration element and accumulation and stress resistance of plant.

Abiotic stress, such as arid, salt marsh, extreme temperature, chemical contamination and oxygen injury can cause serious harm to growing for plant, extreme loss is caused to crop yield, wherein influence of the arid to crop yield, first place is accounted in many natural adverse circumstances, 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 up to 200 270 ten thousand hectares, the national billion cubic meter of the annual water shortage in irrigation district about 30 receives 350 400 hundred million kilograms of grain 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.

Because the resistance to coercive of plant belongs to quantitative character mostly, existing available germ plasm resource is deficient, and the difficulty for improveing stress tolerance in plants using traditional breeding method is quite big, cultivates really resistance to stress kind just particularly difficult.In recent years, with to plant stress-resistance molecular mechanism research deepen continuously and Protocols in Molecular Biology fast development, degeneration-resistant research is deep into molecular level from physiological level, promotes the development of plant stress-resistance genetic engineering.Corresponding responsing reaction can be produced when plant is being forced, to reduce or eliminate the harm brought to plant.This responsing reaction of plant is one and is related to polygenes, multi signal approach, the complex process of polygenes product.These genes and its expression product can be divided into 3 classes：（1) gene and product of signal cascade amplification system and transcription control are participated in；（2) gene and its expression product directly worked to protection biomembrane and protein；（3) protein related with transhipment to the intake of water and ion.In recent years, carried by transgenic technology Research of the high plant to stress-tolerance ability, and to the crops with stress-tolerance ability, the research of xerophyte and halophytes all achieves significant achievement, stress-related genes and signal transduction system there has also been and further understand (Liu Q.1998. Two transcription factors, the and DREB2 of DREB 1, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought and low temperature responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10: 1391-1406； KANGJY.2002. Arabidopsis basic leucine zipper proteins that mediate stress responsive abscisic acid signaling. Plant Cell, 14: 343- 357； ABEH.2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2(MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 15 : 63-78. ) .

But for current research situation, because its mechanism is sufficiently complex, many plants still need to be studied to the biochemistry and physiological response mechanism of adverse circumstance.To be that the contact between degeneration-resistant related signaling pathways and the research of whole signal transmission network system provide important basis in the research in terms of the function and expression regulation of degeneration-resistant response gene.Content of the invention the present inventor utilizes SSH (Subtractive hybridizations）With RACE (cDNA ends rapid amplifyings）The method being combined has cloned a dehydrin protein for cotton（Be named as dehydrin09 herein) encoding gene, and determine its DNA sequence dna.And find to be conducted into after recipient plant and overexpression, the drought tolerance of recipient plant can be significantly improved, and these characters can stablize heredity.

First aspect present invention provides a dehydrin protein dehydrin09 for cotton encoding gene（Ghdehydrin09 is named as herein), its nucleotides sequence is classified as SEQ ID NO: 2.

Second aspect of the present invention provides a kind of recombinant expression carrier, it contains the gene described in first aspect present invention, it is obtained by the way that the gene is inserted into a kind of expression vector, wherein the nucleotide sequence of the gene is operably connected with the expression control sequence of the recombinant expression carrier；Preferably, the expression vector is pCAMBIA2300;Preferably, the recombinant expression carrier is the rd29A-Ghdehydrin09-2300 carriers shown in accompanying drawing 2.

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：By first party of the present invention Recombinant expression carrier described in gene or second aspect of the present invention described in face imports plant or plant tissue and makes the gene expression；Preferably, the plant is tobacco.

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 tobacco.

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 tobacco.

Seventh aspect present invention is provided as the protein of the gene code described in first aspect present invention, its amino acid sequence such as SEQ ID NO:Shown in 1.Brief description of the drawings Fig. 1 is the plant expression vector of Ghdehydrin09 genes（Rd29A-Ghdehydrin09-2300 structure flow)（Scheme l a-lb).

Fig. 2 is the plant expression vector of Ghdehydrin09 genes（Rd29A-Ghdehydrin09-2300 plasmid figure).

Fig. 3 is the T of G//z 7^ genes_QFor transgenic tobacco plant（Middle figure, T_Q6;Right figure, T₀9) and be used as control non-transgenic tobacco plants（Left figure, CK) drought-enduring simulated experiment（Transformation bud transplant 15 days after seedling, arid 14 days（Do not water）, 25 ' C, optical culture/14 hour light culture circulation in 10 hours）As a result.

Fig. 4 is to T using reverse transcription PCR_QFor in transgenic tobacco plant and non-transgenic reference plant

The result of Molecular Detection of G//z ＆ ^9 genes on transcriptional level.M is Marker, and 1-3 is non-transgenic reference tobacco plant, and 4-6 is the inapparent transgene tobacco T of drought-enduring effect_QFor plant, 7-14 is the significant transgene tobacco T of drought-enduring effect_QFor plant.The present invention is further described with reference to non-limiting example for embodiment.

The restriction enzyme in the unreceipted source mentioned in example below is purchased from New England Biolabs companies. Cotton SSH library constructions under the drought stress of embodiment 1：

Specific method is：

SSH libraries are built by Subtractive hybridization method according to the method shown in the PCR-select cDNA Subtraction Kit kit specifications of Clontech companies（Subtracted library）.MRNA using the blade of the cotton seedling of Osmotic treatment in experimentation is used as sample（), Tester the mRNA using the blade of untreated cotton seedling is used as control（Driver).Comprise the following steps that：

(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, illumination cultivation（Photoperiod 16h illumination/8h is dark）, 1/2MS fluid nutrient mediums are poured weekly（Containing 9.39 mM KN0₃, 0.625 mM KH₂P0₄, 10.3 mM H₄N0₃, 0.75 mM MgSO₄, 1.5 mM CaCl₂, 50 μ Μ Κ Ι, 100 μ Μ Η₃ΒΟ₃, 100 M MnSO₄, 30 M ZnS0₄, 1 μ Μ Ν α₂Μο0₄, 0.1 M CoCl₂, 100 μ Μ Na₂EDTA, 100 M FeS0₄, surplus is water）Once.It is used to test when seedling plant height reaches 25-30cm.

(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, cultivates, is normally poured with 1/2MS fluid nutrient mediums under 25 °C, photoperiod 16h illumination/8h dark conditions.Second group is Osmotic treatment group, 25 °C, cultivate under photoperiod 16h illumination/8h dark conditions, stops pouring, handles 10 days, then the blade of timely two groups of seedling apicals 1/3 of clip, after liquid nitrogen quick freeze, is preserved in -70 °C of refrigerators.

(3) Total RNAs extraction：

Control group and each 0.5 g of the cotton leaf of Osmotic treatment group are taken respectively, and the total serum IgE of cotton is extracted with plant RNA extraction kit (Invitrogen).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, detects the integrality of total serum IgE with 1.0% agarose gel electrophoresis, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.MRNAo is separated using the Oligotex mRNA purification kits (purification of poly A+ RNA from total RNA) of Qiagen companies

(4) Subtractive hybridization：

As shown in the PCR-select cDNA Subtraction Kit kit specifications of Clontech companies Method 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 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 the cDNA h of Rsa I digestions 1.5 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) (Unigene) validity, it is to avoid gene is without restriction enzyme site and obtained sequence in non-translational region.In addition to Rsal enzymes, this experiment carries out digestion to Tester cDNA and Driver cDNA by above-mentioned steps with restriction endonuclease Haelll simultaneously and carries out subtractive hybridization and PCR amplifications.Finally merge second of two groups of positive subtractive hybridization cDNA fragments

PCR primer.

(5) structure of subtracted library and preliminary screening, clone and 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 amplification product of cDNA fragments（QIAquick PCR Purification Kit are purified, purchased from Qiagen) it is connected with pGEM-T Easy carriers, comprise the following steps that：Following ingredients are sequentially added in 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.Then, take Ι Ο μ coupled reaction products, it is added in 100 μ L competence e. coli jm109s (being purchased from TAKARA) and mixes, the min of ice bath 30,42 °C of s of heat shock 60, the min of ice bath 2, separately adding 250 μ L LB nutrient solutions, (1% Tryptone is purchased from OXOID, 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 the min of 225 r/min shaken cultivations 30, take 200 μ bacterium solutions to be coated on containing 50 g/mL ampicillins（Purchased from Amresco), 40 g/mL 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）On solid culture flat board, 37 °C of 18 h of cultivation.Count diameter in culture plate>L mm clear white and blue colonies number, random 360 white colonies of picking (numbering：Gh-D001 to Gh-D360).All white colonies are chosen in containing 50 μ_§In 96 porocyte culture plates (CORNING) of the LB fluid nutrient mediums of/η Λ ampicillins, glycerol adding is to final concentration 20% after 37 °C of overnight incubations, then in -80 °C of guarantors Deposit standby.With nest-type PRC bow I things Primer 1 and Primer 2R, (the PCR-selectTM cDNA Subtraction Kit kits of Clontech companies are carried）Enter performing PCR amplification checking to the bacterium solution obtained by the culture, obtain 238 positive colonies, Invitrogen is sent by all positive colonies（Shanghai）The cDNA sequencing analysis of (6) differential cloning are sequenced in trade Co., Ltd：

After the cDNA that DNA sequencing result is removed to carrier and indefinite sequence and redundancy, 134 ESTs are obtained（Expressed sequence tag, EST) (Unigene).Find that wherein 79 Unigene have homologous sequence in GenBank through BlastN（Albumen homology more than 50%）, 30 EST Unknown Functions or for assume albumen, separately there are 25 not obtain homologous matching, thus it is speculated that to be probably the shorter sequence in 3 ' or 5' ends non-translational region.The cotton dehydrin protein gene dehydrin09 of embodiment 2 clone

The sequencing result for numbering the positive colony for being Gh-D222 is removed after redundant DNA, sequence is SEQ ID No:3, sequence analysis shows that the protein of the sequential coding belongs to dehydrin protein, herein by SEQ ID No:3 corresponding total length encoding genes are named as Ghdehydrin09, and its corresponding albumen is named as dehydrin09.

SEQ ID No: 3

1 CAGCTCTTCA AGCGACGAGG AAGAAGGTGA AGGAGAAGAG AAGAAGAAGA AGAAAAAGAA

61 GGAGAAGAAG GGACTGGAAG AAAAGATTGA AGAGAAATTA GAAGGGGAAA AGAAAGAAGA

121 GGACACCTCA GTTCCAGTAG AGAAGTGCGA TGAGCCAGTA GTCCAGCCAG AGACGCCAGA 181 GAAGAAAGGT TTCCTCGAGA AGATCAAGGA GAAACTCCCT GGACAACACA AGAAAGCTGA 241 AGAGGCCAGT AGCCCGGCTC CAGCCCCAGC AGCTGAGCCC CATCACGAAG AGGAGTCAAA 301 GGAAAAGAAG GGAATTTTGG AGAAAATCAA GGAGAAGCTT CCTGGTTACC ACTCCAAATC 361 AGATGAAGAA AAAGAAAAGG CTTGAGATTC AAGTTCACAT AAAAATACTG ATGGGATTGT 421 GTGTTTCTTA AATTGTTCAA AAATTATCTG TTTTTGTTTT TGAGGTTGTA TTTGTTGTAT 481 ATTTCATATT TAAGTTTCTT CTTTTCCCAG CATTTTTAGT ACAAATCATA AGGAAAAAAA 541 AAAGTTTGCT TT

The clone of Ghdehydrin09 total length encoding genes

The Ghdehydrin09 genetic fragments obtained（ SEQ ID No:3) there is terminator codon TGA in, therefore only needed to be 5 ' RACE to clone its total length encoding gene.

According to the SEQ ID No obtained:3 sequences, design three specific primers, and I things and 5'RACE 3 ' end primers are bent as reverse transcription. Ghdehydrin09 GSP 1： SEQ ID NO: 4：

TATGATTTGT ACTAAAAATG

Ghdehydrin09 GSP2： SEQ ID NO: 5：

TCTTGTGTTG TCCAGGGAGT TTC

Ghdehydrin09 GSP3： SEQ ID NO: 6：

TCTGGCGTCT CTGGCTGGAC

Experimental procedure is operated by kit specification（5'RACE System for Rapid Amplification of cDNA Ends kits are purchased from Invitrogen companies）.

CDNA (reverse transcription primer SEQ ID NO are obtained with cotton mRNA reverse transcriptions:4), then add 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:5 (kit is carried with universal primer AAP）, comprise the following steps that：

50 μ PCR reaction systems：5 μ Ι Ο Ε χ Buffer, the mM of 3 μ 2.5 dNTP, 2.0 μm of RNA reverse transcriptions simultaneously add the cDNA after Poly C tails, 1.0 μ Ex Taq (being purchased from TAKARA), 10 μ Μ primer SEQ ID NO:Each 2.0 μ 1 of 5 and AAP, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 55 °C of annealing 30 s, 72 °C of extension 2min), 72 °C of 10 min of extension.

2.0 μ are taken as template after the PCR primer of gained is diluted into 50 times with distilled water, with SEQ ID NO:6 carry out second with universal primer AUAP takes turns PCR amplifications, comprises the following steps that：The Ρ Ο reaction systems of 50 μ 1：5 μ Ι Ο Ε χ Buffer, the mM of 3 μ 2.5 dNTP, the first round PCR primer of 2.0 μ dilutions, the μ Μ of 1.0 μ, 1 Ε χ Τ α 10 primer SEQ ID NO:Each 2.0 μ 1 of 6 and AUAP, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 2 min of extension), 72 °C of 10 min of extension.The band that fragment is about 500 bp is reclaimed from the second wheel PCR primer（Gel Extraction Kit are purchased from OMEGA) pGEM-T Easy carriers are connected to, then it is transformed into escherichia coli jm109 competent cell（Specific method is ibid), and the bacterium solution after conversion is coated on the LB solid mediums containing 50 g/mL ampicillins screened..Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/mL ampicillins respectively to be cultivated, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup. SEQ ID NO:6 carry out bacterium solution PCR amplifications with universal primer AUAP（Reaction system and reaction condition are ibid）, obtain 3 Individual positive colony, send Invitrogen（Shanghai）Trade Co., Ltd is sequenced, and obtains cDNA 5 ' the mountains of the gene by sequence obtained by positive colony sequencing in the 5'RACE products of gained and SEQ ID No:3 splicings, obtain SEQ ID NO: 17：

1 GCGTTCCATT CCATTTCCGT ATAGTTCGAA AACACTTAAG TACCTTCGAT CATATTTTAG

61 TGTCAAAAAA TGGCCGAGGA GCATACCAGC AAGGCCCCTG AGTTGGAGAG CAACGTTAGC

121 GGTGAAGGAG CAGTGGAGAG CAAGGAGCGA GGGTTGTTCG ATTTCATGGG GAAGAAAGAA

181 GAGGAGAAGC CTCAAGAGGA GGTGATCGTA ACCGAGTTTG AAAAGGTTAA TATCGAAGAG

241 ACAAAGGCAG AGGAAGAAGG TGAGGAGAAG AAGAAGCATG GTCTCCTGGA GAAGCTTCAC 301 CGATCAGATA GCAGCAGCTC CAGCTCTTCA AGCGACGAGG AAGAAGGTGA AGGAGAAGAG

361 AAGAAGAAGA AGAAAAAGAA GGAGAAGAAG GGACTGGAAG AAAAGATTGA AGAGAAATTA

421 GAAGGGGAAA AGAAAGAAGA GGACACCTCA GTTCCAGTAG AGAAGTGCGA TGAGCCAGTA

481 GTCCAGCCAG AGACGCCAGA GAAGAAAGGT TTCCTCGAGA AGATCAAGGA GAAACTCCCT

541 GGACAACACA AGAAAGCTGA AGAGGCCAGT AGCCCGGCTC CAGCCCCAGC AGCTGAGCCC 601 CATCACGAAG AGGAGTCAAA GGAAAAGAAG GGAATTTTGG AGAAAATCAA GGAGAAGCTT

661 CCTGGTTACC ACTCCAAATC AGATGAAGAA AAAGAAAAGG CTTGAGATTC AAGTTCACAT

721 AAAAATACTG ATGGGATTGT GTGTTTCTTA AATTGTTCAA AAATTATCTG TTTTTGTTTT

781 TGAGGTTGTA TTTGTTGTAT ATTTCATATT TAAGTTTCTT CTTTTCCCAG CATTTTTAGT

841 ACAAATCATA AGGAAAAAAA AAAGTTTGCT TT

According to SEQ ID NO:17 sequences Design pair of primers are as follows：

Ghdehydrin09 ： SEQ ID NO: 7：

ATGGCCGAGGAGCATACCAGC

Ghdehydrin09R: SEQ ID NO: 8：

TCAAGCCTTTTCTTTTTCTTCATC

Pass through SEQ ID NO:7 and SEQ ID NO:8 clone Ghdehydrin09 complete encoding sequences.Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of cotton.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 2.0 μ cDNA, 1.0 μ PrimeSTAR^ 10 μ Μ primer SEQ ID NO:7 and SEQ ID NO:8 each 2.0 μ 1, and 30 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C are denatured 30 s, 58.C annealing 30 s, 72 °C of extension 2min), 72 °C of 10 min of extension.

Pcr amplification product adds A tails：PCR primer adds 2.5 times of absolute ethyl alcohol, and -20 °C are placed 10 minutes, and centrifugation is removed supernatant, dried, and is dissolved with 21 μ distilled waters.Add 2.5 μ Ι Ο χ Ε χ Buffer, 0.5 μ 5 mM dATP, l ^ Ex Taq.Reaction condition：70 °C are reacted 30 minutes.The DNA fragments for obtaining about 600 bp are reclaimed（Omega QIAquick Gel Extraction Kits）, and be connected on pGEM T-easy carriers（Obtain Ghdehydrin09-pGEM plasmids）, then it is transformed into escherichia coli jm109 competent cell and is screened that (method is ibid on the LB solid mediums containing 50 g/mL ampicillins）.Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/mL ampicillins respectively to be cultivated, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup. SEQ ID NO:7 and SEQ ID NO:8 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 4 positive colonies are obtained, Invitrogen is delivered to（Shanghai）Trade Co., Ltd is sequenced, and gained sequence is SEQ ID NO:2, the amino acid sequence such as SEQ ID NO of its dehydrin09 albumen encoded:Shown in 1.

The amino acid sequence of dehydrin09 albumen： SEQ ID NO: 1

1 MAEEHTSKAP ELESNVSGEG

21 AVESKERGLF DFMGKKEEEK

41 PQEEVIVTEF EKVNIEETKA

61 EEEGEEKKKH GLLEKLHRSD

81 SSSSSSSSDE EEGEGEEKKK

101 KKKKEKKGLE EKIEEKLEGE

121 KKEEDTSVPV EKCDEPVVQP

141 ETPEKKGFLE KIKEKLPGQH

161 KKAEEASSPA PAPAAEPHHE

181 EESKEKKGIL EKIKEKLPGY

201 HSKSDEEKEK A*

The nucleotide sequence of Ghdehydrin09 encoding genes： SEQ ID NO: 2

1 ATGGCCGAGG AGCATACCAG CAAGGCCCCT GAGTTGGAGA GCAACGTTAG CGGTGAAGGA

61 GCAGTGGAGA GCAAGGAGCG AGGGTTGTTC GATTTCATGG GGAAGAAAGA AGAGGAGAAG

121 CCTCAAGAGG AGGTGATCGT AACCGAGTTT GAAAAGGTTA ATATCGAAGA GACAAAGGCA

181 GAGGAAGAAG GTGAGGAGAA GAAGAAGCAT GGTCTCCTGG AGAAGCTTCA CCGATCAGAT

241 AGCAGCAGCT CCAGCTCTTC AAGCGACGAG GAAGAAGGTG AAGGAGAAGA GAAGAAGAAG

301 AAGAAAAAGA AGGAGAAGAA GGGACTGGAA GAAAAGATTG AAGAGAAATT AGAAGGGGAA

361 AAGAAAGAAG AGGACACCTC AGTTCCAGTA GAGAAGTGCG ATGAGCCAGT AGTCCAGCCA

421 GAGACGCCAG AGAAGAAAGG TTTCCTCGAG AAGATCAAGG AGAAACTCCC TGGACAACAC

481 AAGAAAGCTG AAGAGGCCAG TAGCCCGGCT CCAGCCCCAG CAGCTGAGCC CCATCACGAA

541 GAGGAGTCAA AGGAAAAGAA GGGAATTTTG GAGAAAATCA AGGAGAAGCT TCCTGGTTAC

601 CACTCCAAAT CAGATGAAGA AAAAGAAAAG GCTTGA

The Ghdehydrin09 gene plant expression vector establishments of embodiment 3

Plant binary expression vector pCAMBIA2300 is selected (to be purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）As plant expression vector, replace Ρ Τ Π genes with Pnos promoters and contain double enhancers 35S promoter, to reduce expression of the Ρ Τ Π albumen in plant.Inducible promoter rd29A and Tnos terminator are selected respectively as the promoter and terminator of Gfrdefrydrm09 genes, flow is built as shown in Fig. 1.

Use primer SEQ ID NO:9 and SEQ ID NO:10 (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 μ PCR reaction systems： 10 μΐ 5 ><PS Buffer, the mM of 3 μ 2.5 dNTP, the Ρ Β Ι 121 of 1.0 μ 1,1.0 μ Prime STAR, 10 μ Μ primer SEQ ID NO:9 P SEQ ID NO:10 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5, (94 °C of denaturation 30 s, 56 °C of 30 s of annealing, s), 72 °C extend 10 min for 72 °C of extensions 30 for 33 circulations.PCAMBIA2300 (Promega, T4 ligase boxes are connected to as the PCR primer by obtained by after EcoRI, Bglll digestion）Obtain pCAMBIA2300-l.

SEQ ID NO: 9 ：

GCkCGAA rraVTACAAATGGACGAACGGAT

SEQ ID NO: 10：

A CCAGA 7tTAGATCCGGTGCAGATTATTTG

Use primer SEQ ID NO:11 and SEQ ID NO:12 using PBI121 as template amplification Tnos, 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 PBI121,1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:11 and SEQ ID NO:12 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of 30 s of annealing, s), 72 °C extend 10 min for 72 °C of extensions 30.Lj pCAMBIA2300-l (Promega, T4 ligase boxes are connected to as the PCR primer by obtained by after Sacl, EcoRI digestion）Obtain pCAMBIA2300-2

SEQ ID NO: 11：

AAGdCTTGAATTTCCCCGATCGTTCAAA

SEQ ID NO: 12：

CAGAA mCCAGTGAATTCCCGATCTAGTA

Use primer SEQ ID NO:13 and SEQ ID NO:14 with arabidopsis（Colombia's type, purchased from www.arabidopsis.org) DNA be template amplification arabidopsis rd29A promoters（With reference to Zeng J., et L. 2002, Preparation of total DNA from " recalcit rant plant taxa ", Acta Bot. Sin., 44 (6): Method in 694-697 extracts arabidopsis DNA).Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 μ 5 PS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ arabidopsis DNA, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:13 and SEQ ID NO:14 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5, (94 °C of denaturation 30 s, 58 °C of 30 s of annealing, s), 72 °C extend 10 min for 72 °C of extensions 30 for 33 circulations.It is connected to as the PCR primer by obtained by after HindIII, Pstl digestion（Connection method is ibid）PCAMBIA2300-2 obtains pCAMBIA2300-3

SEQ ID NO: 13：

ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO: 14：

TGAd 6TCCAAAGATTTTTTTCTTTCCAATAG

Use bow I thing SEQ ID NO：15 and SEQ ID NO:16 expand positive Ghdehydrin09-pGEM plasmids that Ghdehydrin09 obtained using embodiment 2 as template by PCR）, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 μ 5 xPS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ Ghdehydrin09-pGEM plasmids, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:15 and SEQ ID NO:16 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min), 72 °C of 10 min of extension.It is connected to that (connection method is ibid as the PCR primer by obtained by after Pstl, Sacl digestion）PCAMBIA2300-3, obtains plant expression vector rd29A- Ghdehydrin09-2300 (Fig. 2).

SEQ ID NO: 15：

TGA Cf^ TGGCCGAGGAGCATACCAGC

SEQ ID NO: 16：

The rd29 A-Ghdehydrin09-2300 expression vectors of AAGdCTCTCAAGCCTTTTCTTTTTCTTCATC embodiments 4 convert Agrobacterium

It is prepared by Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competence：Agrobacterium LBA4404 is drawn to single spot inoculation on the LB solid mediums containing 50 g/ml rifampins and 50 g/ml streptomysins, 28 °C are cultivated 1 to 2 day.Picking single bacterium colony is inoculated in 5 ml containing 50 g/ml rifampins and 50 g/ml Overnight incubation (about 12-16 h) is shaken in the LB fluid nutrient mediums of streptomysin, under 28 °C to OD_6(K)It is worth for 0.4, formation seed bacterium solution.Take the seed bacterium solution after 5 ml activation（1 :20 ratio）100 ml are inoculated in containing 50 g/ml rifampins and 50 μ_§In the LB fluid nutrient mediums of the streptomysins of/ι η 1,28 °C are shaken culture 2-2.5 h to OD_6(K)=0.8.The min of ice bath bacterium solution 10, shakes up once every 3 min, makes bacterium even into resting state.10 min are centrifuged in 4000 g under 4 °C, supernatant is abandoned；Add 4000 g under 10% glycerine resuspension thalline of a certain amount of ice precooling, 4 °C and centrifuge 10 min, collect precipitation；Ice-cold 10% glycerine repeats to wash 3-4 times；Adding 10% glycerine of appropriate ice bath precooling, suspended bacterial is precipitated again, that is, LBA4404 competent cells are made, is dispensed, is saved backup in -70 °C with 40 μ/pipe.

Convert Agrobacterium：Melting LBA4404 competent cells on ice, the positive rd29A-Ghdehydrin09-2300 plasmids of gained in 1 μ embodiments 3, the min of ice bath about 10 after mixing are added into the 40 μ competent cell.The mixture of the competent cell after ice bath and positive rd29A-Ghdehydrin09-2300 plasmids is transferred in the electric shock of ice precooling cup with micropipettor, 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 0.1cm electric shock cup, MicroPulser (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 1 ml, 28 °C of preheatings.Quickly and gently mixture is beaten with micropipettor.Suspension is transferred to 1.5 ml centrifuge tube, 28 °C, 225 rpm cultivate 1 h.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 μ_§The streptomysins of/ι η 1,50 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 obtains transgene tobacco using Agrobacterium-medialed transformation method

With 75% alcohol-pickled tobacco seed（Countries tobacco mid-term storehouse, obtains unit：Tobacco institute of the Chinese Academy of Agricultural Sciences, storehouse numbering I5A00660) 30 s, are washed twice with sterilizing distilled water.8 min are soaked with 0.1% mercuric chloride again, is washed with sterilizing distilled water twice, completes surface sterilizing.The tobacco seed of surface sterilizing is placed in MS solid mediums（Containing 18.78 mM KN0₃, 1.25 mM KH₂P0₄, 20.6 mM H₄N0₃, 1.5 mM MgS0₄, 3.0 mM CaCl₂, 50 μΜ ΚΙ, 100 μΜ Η₃ΒΟ₃, 100 M MnSO₄, 30 M ZnSO₄, 1 μ Μ Na₂Mo0₄, 0.1 M CoCl₂, 100 μ Μ Ν α₂ΕϋΤΑ, 100 M FeSO₄, 7.4 g/L agar, the g/L of sucrose 30, surplus is water）On, in being germinateed under aseptic condition, prepare aseptic seedling.Tests for sterility is taken to cut Into the leaf dish of 5 mmx5 mm sizes, the bacterium solution cloned with the Agrobacterium LBA4404 positive transformants of the rd29A-Ghdehydrin09-2300 containing expression vector of gained in the embodiment 4 in exponential phase contaminates the min of leaf dish 10, bacterium solution is blotted, is co-cultured 2 days under dark condition（MS culture mediums）.Blade is gone into differential medium（The MS+1 mg/L basic elements of cell division（BA)+0.1 mg/L methyl α-naphthyl acetates（NAA) the mg/L cephalosporins of+50 mg/L kanamycins+500）On, cultivated 45 days or so under illumination condition, cut after bud is grown up and be transferred to root media（The mg/L cephalosporins of MS+50 mg/L kanamycins+500）Middle culture 30 days or so, preservation is numbered after being transferred to seedling after well developed root system on the MS culture mediums containing 500 mg/L cephalosporins.

Take the transgenic tobacco leaf of acquisition, extract DNA (arabidopsis DNA extraction methods in be the same as Example 3）, with SEQ ID NO:7 and SEQ ID NO:8 (50 μ PCR reaction systems：5 μ Ι Ο χ Ε χ Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ DNA, 1.0 μ Ι Ε χ Τ, 10 μ Μ primer SEQ ID NO:9 standing grain mouthful SEQ ID NO:10 each 2.0 μ 1, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 2 min of extension), 72 °C of 10 min of extension, and the PCR plant for being accredited as the positive are numbered（T_Q1- T_Q20) and preserve.Embodiment 6 is overexpressed Ghdehydrin09 T_QDrought-enduring simulated experiment and Function Identification for transgene tobacco

Sterilized vermiculite is impregnated with 1/2MS fluid nutrient mediums.By the T of the gained of embodiment 5_Q1-T_Q20 transgene tobaccos and control tobacco（Non-transgenic）Tissue-cultured seedling is transplanted to vermiculite respectively, 25 °C, the circulation of the h light cultures of 10 h optical cultures/14, a 1/2MS fluid nutrient medium is poured within every 5 days, strong seedling culture carries out drought stress experiment after 15 days, transgene tobacco and control tobacco arid (are not watered for 14 days）, 25 °C, 10 h optical cultures/14 h light cultures circulations. T_QShow for the Identification of Drought of transfer-gen plant, adjoining tree is all wilted seriously, and T_Q1、 T₀3、 Τ₀5、 Τ₀6、 Τ₀8、 Τ₀9、 Τ₀12、 Τ.15 8 transfer-gen plants can normal growth, show significant drought tolerance（Referring to Fig. 3, with T_Q6、 T_QExemplified by 9, remaining is not shown）.Embodiment 7 verifies G/ on transcriptional level<The expression of fe/z ＆ ^9 genes takes control tobacco, not significantly drought-enduring transgene tobacco T respectively_QFor plant, significantly drought-enduring transgene tobacco To for plant（Upgrowth situation is good）Arid each 0.05 g of blade of 14 days, uses plant RNA extraction kit（Invitrogen) the total serum IgE extracted.Gained 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.According to Invitrogen reverse transcriptions try method shown in neat Ll boxes Superscript III Reverse Transcriptase and carry out reverse transcription（2 μ g total serum IgEs are used as template, reverse transcription primer SEQ ID NO: 8).Pass through SEQ ID NO:7 and SEQ ID NO:8 amplification Ghdehydrin09, detect dehydrin09 albumen relative expression's situations.

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA obtained by above-mentioned reverse transcription.50 μ PCR reaction systems：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:7 and SEQ ID NO:8 each 2.0 μ 1, and 30 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,29 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension lmin), 72 °C of 10 min of extension.

Product electrophoresis result is as shown in Figure 4：M is DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Bioisystech Co., Ltd）, 1-3 is non-transgenic reference tobacco, and 4-6 is the inapparent transgene tobacco T of drought-enduring effect_QFor plant, 7-14 is the significant transgene tobacco T of drought-enduring effect_QFor plant（It is followed successively by： Τ₀1、 Τ。3、 Τ。5、 Τ。6、 Τ。8、 T₀9、 T₀12、 T₀15 ).Stripe size shown in figure and Ghdehydrin09's is in the same size（About 600bp).As a result show there is no the signal of external source G//z/n 7i^ genetic transcriptions, the drought-enduring significant transgene tobacco T of effect in control tobacco_QTranscription for external source G/^e/y^r w^ genes in plant is stronger, the drought-enduring inapparent transgene tobacco T of effect_QTranscribe very weak for plant or do not transcribe.

Claims

Claims

1. a dehydrin protein for cotton, its sequence is SEQ ID N0: 1.

2. encoding the gene of the dehydrin protein of claim 1, its sequence is SEQ ID NO: 2.

3. a kind of recombinant expression carrier, it is obtained by the way that the gene described in claim 2 is inserted into a kind of expression vector, and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector, preferably, the expression vector is pCAMBIA2300.

4. the carrier described in claim 3, it is the rd29A-Ghdehydrin09-2300 carriers shown in accompanying drawing 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 claim 3 or 4 described in claim 2 is imported into plant or plant tissue and makes the gene expression;Preferably, the plant is tobacco.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 tobacco.

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 and the purposes for plant breeding.

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