CN105452275A

CN105452275A - Thellungiella halophila dehydrin protein DH6, coding gene of same, and application thereof

Info

Publication number: CN105452275A
Application number: CN201380078593.7A
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: 2013-09-27
Filing date: 2013-09-27
Publication date: 2016-03-30
Also published as: WO2015042750A1

Abstract

Disclosed are a Thellungiella halophila-sourced dehydrin protein ThDH6, a coding gene of same, and an application thereof in cultivating a transgenic plant of increased drought tolerance.

Description

Thellungiella halophila dehydrin protein DH6, coding gene of same, and application thereof

The present invention relates to vegetable protein and its encoding gene and application with applied technical field for the small salt mustard dehydrin protein of kind and its encoding gene, more particularly to one dehydrin protein and its encoding gene from small salt mustard, 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, is the bottleneck of many regional agricultural developments.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 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 and physiological responses mechanism of adverse circumstance.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.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 small salt mustard（D encoding gene is named as herein, and determines its DNA sequence.And it was found that being conducted into after plant overexpression, it can obviously improve the drought tolerance of transfer-gen plant, and these Character can stablize heredity.

First aspect present invention provides a dehydrin protein D for small salt mustard encoding gene（It is named as herein

ThDH6), 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 35S-7 shown in accompanying drawing 2) H6-2300 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.Brief description of the drawings Fig. 1 be T DH6 plant expression vector C35S-7) H6-2300) and structure flow（Scheme la-lb).

Fig. 2 is T DH6 plant expression vector C35S-7) H6-2300) plasmid figure.

Fig. 3 be 7) H6 T1 for transgenic Arabidopsis plants（In figure, T1F6) and it is used as the non-transgenic Arabidopsis plant of control（In figure, CK) drought-enduring simulated experiment result.（Fig. 3 a are the normal growth Arabidopsis plant of 20 days；Fig. 3 b are normal growth Osmotic treatment Arabidopsis plant of 14 days after 20 days）.

Fig. 4 is protein expression the results of the transgenosis T1 for Arabidopsis plant and non-transgenic reference plant on transcriptional level.M is DNA Ladder Marker (DL2000, TakaRa), and 1-7 is drought-enduring transgenic arabidopsis T1 for plant（It is followed successively by：T1F1, T1F2, T1F3, T1F4, T1F5, T1F6, T1F7), 8-12 be not drought-enduring transgenic arabidopsis Tl for plant, 13-16 is the control of non-transgenic arabidopsis. 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 mentioned in example below is purchased from New England Biolabs companies.Small salt mustard 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.The mRNA of the blade of the small salt mustard seedling of Osmotic treatment is used as sample using in growth course in an experiment（), tester the mRNA using the blade of untreated small salt mustard seedling is used as control（driver).Comprise the following steps that：

(1) material to be tested：

Small salt mustard（T ellungiella halophila, purchased from inner mongolia Ba Yan Nor City carex meyeriana green plants garden halophytes Breeding Center）, it is seeded on sterilized vermiculite, in 25 °C, 16 hours photoperiods illumination/8 hour dark（The Lx of light intensity 2000-3000) under the conditions of cultivate, 1/2MS culture mediums are poured weekly（ 9.39 mM KN0₃, 0.625 mM KH₂P0₄, 10.3 mM NH₄N0₃ , 0.75 mM MgS0₄, 1.5 mM CaCl₂, 50 μ Μ KI, 100 μ Μ Η₃ΒΟ₃, 100 M MnSO₄, 30 μ Μ ZnS0₄, 1 μΜ Να₂Μο0₄, 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：

2 groups, every group of 4 basins, per 1 plant of basin will be divided into for examination seedling.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 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 the small g of salt mustard blade 0. 1 of Osmotic treatment group are taken respectively, use plant RNA extraction kit（It is purchased from

Invitrogen the total serum IgE of small salt mustard blade) is extracted.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.Use the Oligotex mRNA purification kits of Qiagen companies（Purification of polyA+ RNA from total RNA, purify polyA+ from total serum IgE RNA mRNA) is separated.

(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 the g Driver cDNA of 2 Tester cDNA standing grain P 2 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 products of two kinds of positive subtractives hybridization for the first time are mixed, then second of positive subtractive is carried out with the Driver cDNA that are newly denatured and are 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) explanation, 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 in 200 μ PCR pipes：The positive subtractive of purifying hybridizes the μ 1 of second of PCR primer 3, the μ 1 of 2 χ Τ, 4 ligase buffer solutions 5, the μ 1 of pGEM-T Easy carriers 1, the μ of T4 DNA ligases 1 of cDNA fragments, is stayed overnight in 4 °C of connections.Then 10 coupled reaction products are taken, are added in 100 competence e. coli jm109s (being purchased from TAKARA), the min of ice bath 30,42 °C of s of heat shock 60, the min of ice bath 2 separately add 250 L LB fluid nutrient mediums（Contain 1% tryptone（Tryptone, purchased from OXOID), 0.5% yeast extract（Yeast Extract, purchased from OXOID) 1% NaCl of standing grain P (are purchased from traditional Chinese medicines））It is placed in 37 °C of shaking tables, with the min of 225 r/min shaken cultivations 30,200 μ bacterium solutions are then therefrom taken to be coated on containing 50 g/ml ampicillins, 40 g/mL X-gal, 24 g/mL IPTG (X-gal (the chloro- 3- indoles-β-D- galactosides of 5- bromo- 4）With IPTG (isopropyl-β-D- Thiogalactopyranosides）Purchased from TAKARA) LB solid culture plates on, 37 °C cultivate 18 hours.Count diameter in culture plate>1 mm clear white and blue colonies number, random 198 white colonies of picking (numbering：YLS-001 to YLS-198).The white colony of institute's picking is inoculated in after the LB fluid nutrient mediums containing 50 g/mL ampicillins in 96 porocyte culture plates (CORNING), 37 °C of overnight incubations glycerol adding to the (volume ratio of final glycerol concentration 20% respectively）, saved backup in -80 °C.To the colony clone cultivated with nest-type PRC (primer Primer 1 and Primer 2R, the PCR-select cDNA Subtraction Kit kits from Clontech companies）Bacterium solution PCR amplification checkings are carried out, 166 positive colonies is obtained, then send Invitrogen (Shanghai by all positive colonies）Trade Co., Ltd is sequenced.

(6) the cDNA sequencing analysis of differential cloning：

After the cDNA that DNA sequencing results are removed to carrier and indefinite sequence and redundancy, 123 EST (unigene) are obtained₀There are 22 contigs through analysis, there are 101 single sequences.Find that wherein 53 unigene have a homologous sequence in GenBank through BlastN, 21 EST Unknown Functions or to assume albumen separately there are 27 not obtain homologous matching, thus it is speculated that to be probably the shorter sequence in 3' or 5' ends non-translational region.The salt mustard dehydrin protein encoding gene ThDH6 of embodiment 2 clone

Clone YLS-73 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 dehydrin protein, the corresponding total length encoding genes of clone YLS-73 is named as into 7) H6 herein, its corresponding albumen is named as Z) H6.

SEQ ID NO: 3

1 AAACCCAGTC TCTTTGACAA GCTTCACCGA TCCACCAGCT CTTCTTCCTC TTCAAGCGAT

61 GAAGAAGGTG AGGACGGTGA GAAGAGGAAG AAGAAGAAGG AGAAGAAGAA GACTGTCGAA 121 GGAGAGGATA AAACAGAGGA AGAGAATAAA GGAGTAATGG ACAAGATCAA GGAGAAGTTT 181 CCACACGCAA AGAAAACAGA GGATGATCAT GCACCAGTCG TCACCGGCGT CCCAGAGACG 241 GAGAAGATAG GAATGACCGA GAAGATAAAG GAGAAGCTTC CAGGCCACGG CAAGAAACCT 301 GAGGATTCAC CAGTCGTCGA CACCGCGCCG GTGGTTGAGA CAGCGACGCC AATTACGGCG 361 GAGCATTCGG CAGAGCATCC TGCGGAAAAG AAGGGATTTT TGCAAAAGAT CAAAGAAAAG 421 CTTCCAGGTC ATCACGCCAA GGGCACTGAA GAGATGGAGA AGAAAGAAAA AGAGTCTGAT 481 GCTTAA

The clone of DH6 total length encoding genes

According to the SEQ ID NO obtained:3 sequence analyses: SEQ ID NO:3 be the DH6's of encoding gene 7

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.

YLS-73GSP1 : SEQ ID NO: 4：

GGCGTCGCTGTCTCAACCAC YLS-73GSP2 : SEQ ID NO: 5：

TCGACGACTGGTGAATCCTC YLS-73GSP3 : SEQ ID NO:6:

AAACCCAGTCTCTTTGACAAGC 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 YLS-73GSP1 (SEQ ID NO:4) it is reverse transcription primer, reverse transcription is carried out using the mRNA of the small salt mustard blade extraction of Osmotic treatment group as template, obtain cDNA templates, then Poly C tails are added 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 mM of 3 μ 2.5 dNTP, the cDNA of 2.0 μm of RNA reverse transcriptions, 1.0 μ Ex 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 distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 45s, 60 °C of annealing 45 s, 72 °C of extension lmin), 72 °C of extension 7min.

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 μ PCR reaction systems：The first round PCR primer of the mM of 53 μ of μ Ι Ο Χ Ε χ Buffer 2.5 dNTP, 2.0 μ dilution, the μ Μ of 1.0 μ Ex Taq 10 primer SEQ ID NO:5 standing grain P SEQ ID NO:8 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 45 s, 57 °C of annealing 45 s, 72 °C of 1 min of extension), 72 °C of 10 min of extension.Reclaim in second of PCR primer be about 750bp sizes band（Gel Extraction Kit are purchased from OMEGA), and pGEM-T Easy Vector carriers are connected to, being then transformed into JM109, (specific method is ibid）, random 8 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 ^lmL ampicillins and cultivate respectively, and glycerol adding is to (the volume ratio of final glycerol concentration 20% after 37 °C of overnight incubations）, -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）Checking, obtains 3 positive colonies（01st, 03 and 06), send Invitrogen (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 06 of gained, which obtains sequence,: 9： 1 GGGGGGGGGG ATGCTTTTTA AAATCACCTC AAAAAAGCAA TTAAAATCTC AATTCGAATT

61 TGAAATTTTC TCAGCTTTAT TTAATATTCT CGCTAGTCGA TCAAGAGTAA CAATGGCGGA

121 AGAGTACAAG AACGCTTCGG AGGAGTTCAA GAACGTTCCG GAACACGAGA CCACCCCAAA

181 GATCTCCACC ACGGAGGAAC CATCGGCAGA GGTTAAGGAT CGAGGATTTT TCGATTTCTT

241 GGGGAAGAAG AAAGAGGAAG TGAAACCTCA AGAGACCACG ACGCCACTCG AGTCTGAGTT

301 CGAGCACAAG GCTCAGATCT CGGAACCGCC GGCGTTTGTG GCGAAGCACG AAGAAGAGCA

361 AGAGACGAAG GAGAATAAGC CTACTCTCGT CGAGCAGCTT CACCAGAAAC ACGTGGAGGA

421 AGAACAGAAC AAACCCAGTC TCTTTGACAA GCTTCACCGA TCCACCAGCT CTTCTTCCTC

481 TTCAAGCGAT GAAGAAGGTG AGGACGGTGA GAAGAGGAAG AAGAAGAAGG AGAAGAAGAA

541 GACTGTCGAA GGAGAGGATA AAACAGAGGA AGAGAATAAA GGAGTAATGG ACAAGATCAA

601 GGAGAAGTTT CCACACGCAA AGAAAACAGA GGATGATCAT GCACCAGTCG TCACCGGCGT

661 CCCAGAGACG GAGAAGATAG GAATGACCGA GAAGATAAAG GAGAAGCTTC CAGGCCACGG

The sequence SEQ ID NO that 721 CAAGAAACCT GAGGATTCAC CAGTCGTCGA obtain 5 ' RACE:9, the sequence SEQ ID NO with acquisition:3 splicings, are obtained

SEQ ID NO: 10：

1 GGGGGGGGGG ATGCTTTTTA AAATCACCTC AAAAAAGCAA TTAAAATCTC AATTCGAATT

61 TGAAATTTTC TCAGCTTTAT TTAATATTCT CGCTAGTCGA TCAAGAGTAA CAATGGCGGA

121 AGAGTACAAG AACGCTTCGG AGGAGTTCAA GAACGTTCCG GAACACGAGA CCACCCCAAA

181 GATCTCCACC ACGGAGGAAC CATCGGCAGA GGTTAAGGAT CGAGGATTTT TCGATTTCTT

241 GGGGAAGAAG AAAGAGGAAG TGAAACCTCA AGAGACCACG ACGCCACTCG AGTCTGAGTT

301 CGAGCACAAG GCTCAGATCT CGGAACCGCC GGCGTTTGTG GCGAAGCACG AAGAAGAGCA

361 AGAGACGAAG GAGAATAAGC CTACTCTCGT CGAGCAGCTT CACCAGAAAC ACGTGGAGGA

421 AGAACAGAAC AAACCCAGTC TCTTTGACAA GCTTCACCGA TCCACCAGCT CTTCTTCCTC

481 TTCAAGCGAT GAAGAAGGTG AGGACGGTGA GAAGAGGAAG AAGAAGAAGG AGAAGAAGAA

541 GACTGTCGAA GGAGAGGATA AAACAGAGGA AGAGAATAAA GGAGTAATGG ACAAGATCAA

601 GGAGAAGTTT CCACACGCAA AGAAAACAGA GGATGATCAT GCACCAGTCG TCACCGGCGT

661 CCCAGAGACG GAGAAGATAG GAATGACCGA GAAGATAAAG GAGAAGCTTC CAGGCCACGG

721 CAAGAAACCT GAGGATTCAC CAGTCGTCGA CACCGCGCCG GTGGTTGAGA CAGCGACGCC

781 AATTACGGCG GAGCATTCGG CAGAGCATCC TGCGGAAAAG AAGGGATTTT TGCAAAAGAT

841 CAAAGAAAAG CTTCCAGGTC ATCACGCCAA GGGCACTGAA GAGATGGAGA AGAAAGAAAA

901 AGAGTCTGAT GCTTAA are according to SEQ ID NO:10 sequence analyses, SEQ ID NO:10 be 7 DH6 full length sequence.According to SEQ ID NO:10 sequences Design pair of primers are as follows：

ThDH6F : SEQ ID NO: 1 1：

ATGCTTTTTAAAATCACCTCA

ThDH6R: SEQ ID NO: 12：

TTAAGCATCAGACTCTTTTTC AP : SEQ ID NO: 13：

GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT passes through SEQ ID NO:11 and SEQ ID NO:12 clone 7 DH6 complete encoding sequences. The RNA of the small salt mustard of Osmotic treatment group is extracted as template, with primer SEQ ID NO:13 be reverse transcription primer, and reverse transcription obtains small salt mustard cDNA, then using stratagene PfuUltra II Fusion HS DNA Polymerase, and performing PCR reaction is entered by template of the cDNA of the small salt mustard of above-mentioned acquisition.50 μ PCR reaction systems：The mM of 5 μ, 10 0.5 μ of X PfuUltra II reaction Buffer 25 dNTP, the μ PfuUltra II Fusion HS DNA Polymerase of 2.0 μ cDNA 1.0,10 μ Μ primer SEQ ID NO:11 and SEQ ID NO:12 each 2.0 μ 1, and 37.5 μ distilled water.PCR reaction conditions：95 °C of min of pre-degeneration 2,35 circulations（95 °C of denaturation 25 s, 50 °C of annealing 25 s, 72 °C of extension 40s), 72 °C of 5 min of extension.

Pcr amplification product adds A tails：PCR primer moisturizing removes removing protein one time to 400 μ 1, first with chloroform, draws supernatant and adds the μ 1 of 3 Μ sodium acetate solutions 40, the absolute ethyl alcohol of 2 times of volumes is added, -20 °C are placed 10 minutes, centrifugation, supernatant is removed, is dried, is dissolved with 21 μ distilled waters.Add the mM of 2.5 0.5 μ of the μ Ι Ο Χ Ε χ Buffer 5 μ Ex Taq of dATP standing grain P 1.0.Reaction condition：70 °C are reacted 30 minutes.Obtained about 900bp DNA fragmentation is reclaimed（Omega QIAquick Gel Extraction Kits）, it is connected on pGEM T-easy carriers（Obtain ThDH6-pGEM plasmids）, then convert JM109, random 8 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/mL ampicillins and cultivated respectively, glycerol adding is to (the volume ratio of final glycerol concentration 20% after 37 °C of overnight incubations）, -80 °C save backup.With primer SEQ ID NO:11 and SEQ ID NO:12 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 3 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 DH6 albumen： SEQ ID NO: 1

1 LFKITSKKQ LKSQFEFEIF SALFNILASR SRVTMAEEYK NASEEFK VP EHETTPKIST

61 TEEPSAEVKD RGFFDFLGKK KEEVKPQETT TPLESEFEHK AQISEPPAFV AKHEEEQETK

121 ENKPTLVEQL HQKHVEEEEN KPSLFDKLHR SSSSSSSSSD EEGEDGEKRK KKKEKKKTVE

181 GEDKTEEENK GV DKIKEKF PHAKKTEDDH APWTGVPET EKIG TEKIK EKLPGHGKKP

241 EDSPWDTAP WETATPITA EHSAEHPAEK KGFLEKIKEK LPGHHAKGTE E EKKEKESD

301 A

The nucleotide sequence of 7 DH6 encoding genes： SEQ ID NO: 2

1 ATGCTTTTTA AAATCACCTC AAAAAAGCAA TTAAAATCTC AATTCGAATT TGAAATTTTC

61 TCAGCTTTAT TTAATATTCT CGCTAGTCGA TCAAGAGTAA CAATGGCGGA AGAGTACAAG

121 AACGCTTCGG AGGAGTTCAA GAACGTTCCG GAACACGAGA CCACCCCAAA GATCTCCACC

181 ACGGAGGAAC CATCGGCAGA GGTTAAGGAT CGAGGATTTT TCGATTTCTT GGGGAAGAAG

241 AAAGAGGAAG TGAAACCTCA AGAGACGACG ACGCCACTCG AGTCTGAGTT CGAGCACAAG

301 GCTCAGATCT CGGAACCGCC GGCGTTTGTG GCGAAGCACG AAGAAGAGCA AGAGACGAAG

361 GAGAATAAGC CTACTCTCGT CGAGCAGCTT CACCAGAAAC ACGTGGAGGA AGAAGAGAAC

421 AAACCCAGTC TCTTTGACAA GCTTCACCGA TCCAGCAGCT CTTCTTCCTC TTCAAGCGAT

481 GAAGAAGGTG AGGACGGTGA GAAGAGGAAG AAGAAGAAGG AGAAGAAGAA GACTGTCGAA

541 GGAGAGGATA AAACAGAGGA AGAGAATAAA GGAGTAATGG ACAAGATCAA GGAGAAGTTT 601 CCACACGCAA AGAAAACAGA GGATGATCAT GCACCAGTCG TCACCGGCGT CCCAGAGACG

661 GAGAAGATAG GAATGACCGA GAAGATAAAG GAGAAGCTTC CAGGCCACGG CAAGAAACCT

721 GAGGATTCAC CAGTCGTCGA CACCGCGCCG GTGGTTGAGA CAGCGACGCC AATTACGGCG

781 GAGCATTCGG CAGAGCATCC TGCGGAAAAG AAGGGATTTT TGGAAAAGAT CAAAGAAAAG 841 CTTCCAGGTC ATCACGCCAA GGGCACTGAA GAGATGGAGA AGAAAGAAAA AGAGTCTGAT

The ￡ of 901 GCTTAA embodiments 37>H6 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 CaMV35S promoters that Ν Ρ Τ Π genes contain double enhancers are replaced with Pnos promoters, to reduce expression of the Ν Ρ Τ Π albumen in plant.35S promoter and terminator Tnos are inserted in the upstream of Pnos promoters respectively as the promoter and terminator of 7 DH6 genes, 7 DH6 genes are between the 35S promoter and Tnos terminators.

With primer SEQ ID NO:14 and SEQ ID NO:15 (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：The xPS Buffer of 10 μ 5, the mM of 3 μ 2.5 dNTP, the μ PrimeSTAR HS archaeal dna polymerases of 1.0 μ pBI121 1.0,10 μ Μ primer SEQ ID NO:14 and SEQ ID NO:15 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, 56 °C of 30 s of annealing, s), 72 °C extend 10 min for 72 °C of extensions 30.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: 14 ：

GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 15 :

ATCCAGATCTAGATCCGGTGCAGATTATTTG uses primer SEQ ID NO:16 and SEQ ID NO:17 using pBI121 as template amplification Tnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：The mM of 10 μ, 5 xPS Buffer, 3 μ 2.5 dNTP, 1.0 μ pBI121,1.0 μ PrimeSTAR HS archaeal dna polymerases, 10 μ Μ primer SEQ ID NO:16 and SEQ ID NO:17 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.PCAMBIA2300-l (Promega T4 ligase boxes are connected to as the PCR primer by obtained by after Kpnl, EcoRI digestion）Obtain pCAMBIA2300-2.

SEQ ID NO: 16： CGGGG7MCCGAATTTCCCCGATCGTTCAAA

SEQIDNO: 17：

TCKGAA rrCCC AGTGAATTCCCGATCT AGT A use primer SEQIDNO:18 and SEQIDNO:19 using pCAMBIA2300 plasmids as template amplification 35S promoters.Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ Ι Ρ Ο reaction systems：The mM of 10 3 μ of μ 5xPS Buffer 2.5 dNTP, 1.0 μ dilute 50 times of pCAMBIA2300 plasmids, 1.0 μ PrimeSTAR HS archaeal dna polymerases, 10 μ Μ primer SEQIDNO:18 and SEQIDNO:19 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, 50 °C of 30 s of annealing, s), 72 °C extend 10min for 72 °C of extensions 30.It is connected to as the PCR primer by obtained by after HindIII, Xbal digestion（Connection method is ibid）PCAMBIA2300-2 obtains pCAMBIA2300-3

SEQIDNO: 18：

ACT^GCrJATGGTGGAGCACGACACTCT

SEQIDNO: 19：

GC r AG AG AT AG ATTTGT AG AG AG AG AC use primer SEQ ID NO:20 and SEQ ID NO:(template is the positive 7 DH6-pGEM plasmids that embodiment 2 is obtained to 21 amplification ThDH6）, using stratagene PfuUltra II Fusion HS DNA Polymerase.50 μ PCR reaction systems：5 μ lOxPfuUltra II reaction Buffer, the mM of 0.5 μ 125 dNTP, 2.0 μ ThDH6-pGEM plasmids, the μ Μ of 1.0 μ PfuUltra II Fusion HS DNA Polymerase 10 primer SEQ IDNO:20 and SEQIDNO:21 each 2.0 μ 1, and 37.5 μ distilled water.PCR reaction conditions：95 °C of pre-degeneration 2min, 35 circulations（95 °C of denaturation 25 s, 50 °C of annealing 25 s, 72 °C of extension 30s), 72 °C of 5 min of extension.Connected as the PCR primer by obtained by after Xbal, Kpnl digestion（Connection method is ibid）To pCAMBIA2300-3, plant expression vector 35S-7 DH6-2300 are obtained.

SEQIDNO: 20：

AATCTAGAATGCTTTTTAAAATCACCTCA SEQIDNO: 21：

The 35S-ThDH6-2300 expression vectors of GCGGTACCTTAAGCATCAGACTCTTTTTC embodiments 4 convert Agrobacterium

The preparation of Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competent cell：1-2 in advance Agrobacterium LBA4404 is drawn single spot on the LB solid mediums containing 50 g/ml rifampins and 50 g/ml streptomysins and is inoculated with by it, and 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）It is inoculated in LB fluid nutrient mediums of 100 ml containing 50 g/ml rifampins and 50 g/ml streptomysins, 28 °C of shakes cultivate 2-2.5 hours to OD₆..=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 10% glycerine of a certain amount of ice precooling（Volume）Resuspension thalline, 4000 g centrifuge 10 min under 4 °C, collect precipitation；Ice-cold 10% glycerine（Volume）Repetition is washed 3-4 times；Add 10% glycerine of appropriate ice precooling（Volume）Again suspended bacterial is precipitated, that is, LBA4404 competent cells are made, is dispensed, is saved backup in -70 °C with 40 μ/pipe.

Convert Agrobacterium：Melt competent cell on ice, the positive 35S-7 of gained in 1 μ embodiments 3 added into 40 μ competent cell) H6-2300 plasmids, the min of ice bath about 10 after mixing.The mixture of the competent cell and 35S-ThDH6-2300 DNAs is transferred to the electric shock cup of ice precooling with liquid-transfering gun（Purchased from bio-rad) in, rapping makes suspension reach electric shock cup bottom, has been careful not to bubble.Electric shock cup is put on the slideway of electroporation chamber, promotes slideway to put electric shock cup to electroporation chamber base electrode.Using the electric shock cup of 0.1 cm specifications, 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 28 °C of preheatings.It is quickly soft to be beaten competent cell with liquid-transfering gun.Suspension is transferred to 1.5 ml centrifuge tube, 28 °C, 225 rpm shake culture 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 streptomysins and 50 g/ml kanamycins）, 28 °C of cultures.Positive transformants clone is screened, and its bacterium solution is saved backup in -70 °C.

Embodiment 5 obtains transgenic arabidopsis using Agrobacterium-medialed transformation method

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.Before transplanting 1/2MS culture mediums are poured per alms bowl（9.39 mM KN0₃, 0.625 mM KH₂P0₄, 10.3 mM H₄N0₃, 0.75 mM MgS0₄, 1.5 mM CaCl₂, 50 μΜ ΚΙ, 100 μΜ Η₃ΒΟ₃, 100 M MnSO₄, 30 M ZnSO₄, 1 μ Μ Na₂Mo0₄, 0.1 μΜ CoCl₂, 100 μΜ Na₂EDTA, 100 μΜ FeS0₄) 40 ml, transplant backsight soil moisture and keep the skin wet in time.In the appropriate nutrient solution of growth period.On demand per 3-4 weeks once（Or the time is longer）.In order to obtain more bud on each plant, first flower is cut off after most of first inflorescence of plant are formed Sequence, releases apical dominance, promotes the synchronous appearance of multiple secondary inflorescences.When most of inflorescences about 1-10 cm are high（Cut off after first inflorescence about 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）In, 250 r/min shake incubated overnight under 28 °C of constant temperature.Resulting bacterium solution is inoculated into the sterile LB Liquid Cultures of 200 mL by 1%-2% volume ratio again（Containing 75 mg/L rifampins, 100 mg/L streptomysins and 100 mg/L kanamycins）In, 250 171^^ under 28 °C【The temperature that extends shaking makes the concentration of Agrobacterium reach 00₆₍₎₍₎=1.8, then at 4 °.Lower 3000 r/min are centrifuged after 15 min, abandoning supernatant with dip-dye culture medium（The dip-dye culture medium be Jia 5.0% in 1/2MS culture mediums (w/v) sucrose and 0.05% (500 μ Ι 7) 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 3-5 s in agrobacterium suspension, 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 (the 4.695 mM KN0 containing 1/4 MS₃, 0.3125 mM KH₂P0₄, 5.15 mM H4NO3,0.375 mM MgS0₄, 0.75 mM CaCl₂, 25 μΜ ΚΙ, 50 μΜ Η₃ΒΟ₃, 50 M MnSO₄, 15 M ZnS0₄, 0.5 μ Μ Ν α₂Μο0₄, 0.05 M CoCl₂, 50 μ Μ Na₂EDTA, 50 M FeSO4) a great number of elements the aqueous solution, add 0.8 % agar powders, agar is heated to micro-wave oven to dissolve completely, it is to be cooled to 50 °C or so, the desired amount of final concentration of 50 mg kanamycins is added, 25 mL are poured into every culture dish after shaking up, putting can sow after experimental bench cooled and solidified.Load weighted seed is poured on a plain copying 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: 11：Standing grain P SEQ ID NO:12 amplification ThDH6,50 μ PCR reaction systems：5 μ Ι Ο Ε χ Buffer, the mM of 3 μ 2.5 dNTP, 2.0 μ DNA, the μ Μ of 1.0 μ, 1 Ε χ Τ α 10 primer SEQ ID NO:11 and SEQ ID NO:12 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 45 s, 50 °C of 45 s of annealing, s), the PCR plant for being accredited as the positive are numbered 72 °C of 7 min of extension for 72 °C of extensions 45（T1F1-T1F12), And preserve.Embodiment 6 is overexpressed the sterilized vermiculite of drought-enduring simulated experiments and Function Identification of the T DH6 transgenic arabidopsis T1 for plant and is impregnated with 1/2MS culture mediums.T1F1-T1F6 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 4-5 more consistent seedling 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 TO 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 T1F1, T1F2, T1F3, T1F4, T1F5, T1F6 totally 24（Per each 4-5 of strain）20 can survive and continued growth in arabidopsis, show obvious drought tolerance（Referring to Fig. 3 a and 3b, by taking T1F6 as an example, T1F1, T1F2, T1F3, TIF 4, T1F5 result it is similar with T1F6, be not shown here）.Embodiment 7 verifies 7) H6 protein expressions on transcriptional level

Control Arabidopsis plant, drought-enduring transgenic arabidopsis T1 is taken (to be belonging respectively to seven strains of T1F1, T1F2, T1F3, T1F4, T1F5, T1F6, T1F7 for plant respectively）Not drought-enduring transgenic arabidopsis T1 uses plant RNA extraction kit for each 0.05 g of the arid blade of 10 days of plant（Invitrogen total serum IgE) is 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.Method shown in neat Ll boxes Superscript III Reverse Transcriptase, which is tried, according to Invitrogen reverse transcriptions carries out reverse transcription（2 total serum IgEs are as template, and reverse transcription primer is SEQ ID NO: 13 ).Pass through SEQ ID NO:L l and SEQ ID NO:12 amplification T DH6, detect DH6 albumen relative expression's situations.

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：The xPS Buffer of 10 μ 5, the mM of 3 μ 2.5 dNTP, the μ PrimeSTAR HS archaeal dna polymerases of 2.0 μ cDNA 1.0,10 μ Μ primer SEQ ID NO:11 standing grain P SEQ ID NO:12 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 45 s, 50 °C of annealing 45 s, 72 °C of extension 45s), 72 °C of 10 min of extension.

Product electrophoresis result is as shown in Figure 4：M is DNA Ladder Marker (DL2000, TakaRa), and 1-7 is drought-enduring transgenic arabidopsis T1 for plant（It is followed successively by：T1F1, T1F2, T1F3, T1F4, T1F5, T1F6, T1F7), 8-12 be not drought-enduring transgenic arabidopsis Tl for plant, 13-16 is the control of non-transgenic arabidopsis.The size of PCR primer electrophoretic band shown in figure is in the same size with 7 DH6's（About 900bp).As a result show, control arabidopsis does not have ThDH6 transcriptions, and drought-enduring transgenic arabidopsis T1 is stronger for the transcription of ThDH6 in plant, and not drought-enduring transgenic arabidopsis T1 is very weak for the transcription of ThDH6 in plant.

Claims

Claims

1. the albumen of one small salt mustard Dehydrins encoding gene coding, its amino acid sequence is SEQ ID NO: 1.

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-7 DH6-2300 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：By the gene or claim described in claim 2

Recombinant expression carrier described in 3 or 4 imports 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 and the purposes for plant breeding.

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