CN105189537A

CN105189537A - Cotton leucine zipper protein bZIP-3, coding genes of same, and application thereof

Info

Publication number: CN105189537A
Application number: CN201380074537.6A
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: 2013-04-19
Filing date: 2013-04-19
Publication date: 2015-12-23
Also published as: WO2014169472A1

Abstract

The present invention relates to a plant protein, the coding genes of same, and an application thereof, and specifically relates to a leucine zipper protein sourced from cotton, bZIP-3, the coding genes of same, and an application thereof in cultivating a transgenic plant of increased drought tolerance.

Description

Cotton leucine zipper protein bZIP-3, coding genes of same, and application thereof

One grows cotton leucine zipper protein bZIP-3 and its encoding gene and application

The present invention relates to vegetable protein and its encoding gene and application, the more particularly to one leucine zipper protein bZIP-3 and its encoding gene from cotton, and its application in the genetically modified plants that drought tolerance is improved are cultivated for technical field.The environment stresses such as background technology 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 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, high-quality germ plasm resource 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 just current research situation ^ says that, because its mechanism is sufficiently complex, many plants still need to be studied to the biochemistry and physiological responses mechanism of adverse circumstance.To be that the contact between the related signaling pathways of plant stress-resistance 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 leucine zipper protein for cotton（Be named as bZIP-3 herein) encoding gene, and determine its DNA sequence.And it was found that being conducted into after recipient plant and overexpression, the drought tolerance of transfer-gen plant can be significantly improved, and And these characters can stablize heredity.

First aspect present invention provides a leucine zipper protein bZIP-3 for cotton encoding gene（GhbZIP-3 is named as herein), 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 it is obtained by the way that the gene is inserted into a kind of expression vector, it is preferable that the expression vector is PCAMBIA2300;And the nucleotide sequence of the gene is operably connected with the expression control sequence of the recombinant expression carrier；Preferably, the recombinant expression carrier is the 35S-G shown in accompanying drawing 2) Z/P-3-2300 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 the resistance to morning property of plant and the purposes for plant breeding;Preferably, the plant is arabidopsis.

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.It is G to illustrate Fig. 1) the plant expression vector C35S-G of Z/P-3 genes) Z/P-3-2300;>Structure flow（Scheme la-lb).Fig. 2 is the plasmid figure of the plant expression vector (35S-GMZ/P-3-2300) of GhbZIP-3 genes.

Fig. 3 is G) T2 of Z/P-3 genes is for transgenic Arabidopsis plants（In figure, T2F1;T2F5) and be used as control non-transgenic Arabidopsis plant（In figure, CK1, CK2) 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 to utilize Molecular Detection the result of the reverse transcription PCR to T2 for transgenic Arabidopsis plants and non-transgenic reference plant kind GhbZIP-3 genes on transcriptional level.M is DNA Ladder Marker (DL2000, TakaRa), and 1-6 is the notable transgenic arabidopsis T2 of drought-enduring effect for plant（It is followed successively by： T2FK T2F2、 T2F3、 T2F4、 T2F5、 T2F6) ；7-10 is the inapparent transgenic arabidopsis T2 of drought-enduring effect for plant;11-14 is that the present invention is further described with reference to non-limiting example for non-transgenic control Arabidopsis plant embodiments.

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

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 mRNA), control is used as using the mRNA of the blade of untreated cotton seedling（Driver mRNA).Ju Ti Walk are suddenly 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, photoperiod 16h illumination/8h dark（The Lx of light intensity 2000-3000) 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 NH₄N0₃ , 0.75 mM MgSO₄, 1.5 mM CaCl₂, 50 μ Μ Κ Ι, 100 μ Μ Η₃ΒΟ₃, 100 μ Μ MnS0₄, 30 μ Μ ZnS0₄, 1 μΜ Να₂Μο0₄, 0.1 μΜ CoCl₂, 100 μΜ Na₂EDTA, 100 μΜ FeS0₄) once.It is used to test when seedling plant height reaches 25-30 cm.

(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, photoperiod 16h illumination/8h dark conditions, is only often poured.Second group is Osmotic treatment group, 25 °C, cultivate under photoperiod 16h illumination/8h dark conditions, 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 0.5 g of the cotton leaf of Osmotic treatment group are taken respectively, use plant RNA extraction kit（Purchased from Invitrogen) extract cotton leaf total serum IgE.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；With 1.0% agarose gel electrophoresis detect total serum IgE integrality, 28S bands it is bright Degree is about 2 times of 18 S bands, shows that RN A integrality is good.MRNA is separated using the Oligotex mRNA purification kits (purification of poly A+ RNA from total RNA) of Qiagen companies.

(4) Subtractive hybridization：

Method as shown in the PCR- select cDN A Subtraction Kit kit specifications of Clontech companies 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) the validity of (Unigene), avoid gene without restriction enzyme site and obtained sequence in non-translational region, in addition to Rsal enzymes, 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 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 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, pGEM-T Easy carriers 1 μ 1, the μ of T4 DNA ligases 1 of cDNA fragments, is stayed overnight in 4 °C of connections.10 μ coupled reaction products are taken, is added in 100 μ 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 add 250 μ L L 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 the min of 225 r/min shaken cultivations 30, gained bacterium solution is 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 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>1 mm clear white and blue colonies number, random 198 white colonies of picking (are compiled Number：Gh-B001 to Gh-B198).All white colonies are inoculated in 96 porocyte culture plates (CORNING) of the LB fluid nutrient mediums containing 50 g/mL ampicillins respectively, 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 positive colonies is obtained, is then sending Invitrogen by all positive colonies（Shanghai）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, 135 EST (Unigene are obtained；).There are 21 contigs through analysis, there are 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 cotton leucine zipper protein encoding gene G of embodiment 2) Z/P-3 clone

The sequencing result for numbering the positive colony for being Gh-B98 is removed after redundant DNA, sequence is SEQ ID No:3, sequence analysis shows that the protein of the sequential coding belongs to leucine zipper protein, herein by SEQ ID No:3 corresponding total length encoding genes are named as GhbZIP-3, and its corresponding albumen is named as bZIP-3.

SEQ ID No: 3

1 GGTGGTAATC AAAATGGTAT TCAAGCTTCC GATTTGCCTA TGAATTTTAA TGGAGTTAGA

61 TCCAATCAGC ACCACTTAAC TCAGCTTCAG CAGCAACCAT TATGTCCTAA GCAACCTGGT

12 1 GTGGAATATG GAGCTCAGAT GGGTTTGCAA AGTGGTGGTC AGTTGGGGAG TCCTGGAATT

18 1 AGGTGTGGTC AGGGACTATA TAATGGCTTG ATCCATGGTG GTGGGATGAG TATGGTTGGT

24 1 TTAAGATCAC CTGCTAACCA CTTTTCATCA GATGGGATTG GGAAGAGTAG TGGAGATAGT

3 0 1 TCCTCAGTTT CACCCGTTCC TTATGTGTTT AATGGAAGTT TGAGGGGTAG GAAAAGCAGT

36 1 GCTGTGGAAA AGGTTGCTGA GAGGAGGCAG AGAAGAATGA TAAAGAACCG AGAATCCGCT

42 1 GCAAGATCAC GAGCTCGCAA GCAGGCTTAT ACAACGGAGT TGGAAGCAGA AGTTGCTAAG

4 8 1 CTGAAAGAGG AGAACCAAGA ATTGCAGAAG AAACA GA C ACATCATAAA AATACAGAAA

54 1 AATCAGGTGA TGGAGATGAT GAATGTGCAG CCAGGAGCTA AGAAACGATG CTTGCGACGA

60 1 ACACGGACAG GTCCTTGGTG AATC

The clone of GhbZIP-3 total length encoding genes

According to the SEQ ID No obtained:3 sequence analyses: SEQ ID No:3 be encoding gene

3 ' terminal sequences.According to the SEQ ID NO obtained:3 sequences, design following three specific primers, draw as reverse transcription 3 ' end primers of thing and 5 ' RACE.

GhB98 GSP1 : SEQ ID NO: 4：

GACCACCACTTTGCAAACCC

GhB98 GSP2 : SEQ ID NO: 5：

CATATTCCACACCAGGTTGC

GhB98 GSP3 : SEQ ID NO:6:

GGTGGTAATCAAAATGGTATTC

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 GhB98 GSP1 (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 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:4 standing grain mouthful 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 denaturation 30 s, 56 °C of annealing 30 s, 72 °C of extension lmin), 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: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：5 μ Ι Ο Ε χ Buffer, the mM of 3 μ 2.5 dNTP, the first round of 2.0 μ dilutions

PCR primer, 1.0 l Ex Taq, 10 μ Μ 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 min of pre-degeneration 5,33 circulations（94 denaturation 45 s, 56 °C of annealing 45 s, 72V extend 1 min), 72 °C of 10 min of extension.Reclaim the band of about 750 bp sizes in the second wheel PCR primer（Gel Extraction Kit are purchased from OMEGA), and pGEM-T Easy carriers are connected to, then it is transformed into e. coli jm109 competent cell（Specific method is ibid), and the bacterium solution after conversion is coated on containing 50 μ_§Screened on the LB solid mediums of/η Λ 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.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）, obtain 3 positive colonies（Gh9-1, Gh9-5 and Gh9-8), 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 positives clone Gh9-8 sequencings of the 5'RACE products of gained are obtained into sequence: 9：

1 GGGGGGGGCA CATGAACTTC AAGAACTTTG GAAGTAACCA ACCGCCGTCA GGTGATGGCG

61 GCGGTGGTAA TAAACCGCCT GGGAATTATC CGATAAGTAG ACAGCCATCG GTCTATTCTC

12 1 TAACCTTTGA TGAGTTTCAA AGCACAATGG GTGGAATAGG CAAAGATTTC GGGTCGATGA

18 1 ACATGGATGA GCTGTTAAAG AACATTTGGA GTGCTGAAGA AACTCAAACA ATGGCTTCTC

24 1 CCGGTGTTGG CCTAGTGGGA AATGGAGGGT ACAAAGGCA AGGGTCTTTG ACTTTGCCCA

3 0 1 GGACACTTAG CCACAAAACT GTTGATGAAG TTTGGAGAGA GA CTCAAAT GAGTTTTCTA

36 1 TAGGGACTGA AGCCACTGAT AATATGCCAC AAAGACAGCA AACCTTAAAG GAGATTACTT

42 1 TAGAGGAGTT TTTGGTCAGA GCCGGTGTGG TAAGAGAGGA TAACATCCAA TTTTCTGGGA

4 8 1 AGGACAATAA TGTTAATGGC GGCTTCTTTG GGGAATTACC GCAAGCAGGG AGTAA ACCA

54 1 GTGGGTTTGG GATTGGGTTT CAACAAGGTG GAAGAGGTCC AAATTTGATG GGGAATAGGC

60 1 TACCTGATGG TGGTAATCAA AATGGTATTC AAGCTTCCGA TTTGCCTATG AATTTTAATG

66 1 GAGTTAGATC CAATCAGCAC CACTTAACTC AGCTTCAGCA GCAACCATTA TGTCCTAAGC

The sequence SEQ ID NO that 72 1 AACCTGGTGT GGAATATG obtain 5 ' RACE:9, the sequence SEQ ID NO with above-mentioned acquisition:3 splicings, are obtained

SEQ ID NO: 10：

1 GGGGGGGGCA CATGAACTTC AAGAACTTTG GAAGTAACCA ACCGCCGTCA GGTGATGGCG

61 GCGGTGGTAA TAAACCGCCT GGGAATTATC CGATAAGTAG ACAGCCATCG GTCTATTCTC

12 1 TAACCTTTGA TGAGTTTCAA AGCACAATGG GTGGAATAGG CAAAGATTTC GGGTCGATGA

18 1 ACATGGATGA GCTGTTAAAG AACATTTGGA GTGCTGAAGA AACTCAAACA ATGGCTTCTC

24 1 CCGGTGTTGG CCTAGTGGGA AATGGAGGGT TACAAAGGCA AGGGTCTTTG ACTTTGCCCA

3 0 1 GGACACTTAG CCACAAAACT GTTGATGAAG TTTGGAGAGA GATCTCAAAT GAGTTTTCTA

36 1 TAGGGACTGA AGCCACTGAT AATATGCCAC AAAGACAGCA AACCTTAAAG GAGATTACTT

42 1 TAGAGGAGTT TTTGGTCAGA GCCGGTGTGG TAAGAGAGGA TAACATCCAA TTTTCTGGGA

4 8 1 AGGACAATAA TGTTAATGGC GGCTTCTTTG GGGAATTACC GCAAGCAGGG AGTAATACCA

54 1 GTGGGTTTGG GATTGGGTTT CAACAAGGTG GAAGAGGTCC AAATTTGATG GGGAATAGGC

60 1 TACCTGATGG TGGTAATCAA AATGGTATTC AAGCTTCCGA TTTGCCTATG AATTTTAATG

66 1 GAGTTAGATC CAATCAGCAC CACTTAACTC AGCTTCAGCA GCAACCATTA TGTCCTAAGC

72 1 AACCTGGTGT GGAATATGGA GCTCAGATGG GTTTGCAAAG TGGTGGTCAG TTGGGGAGTC

78 1 CTGGAATTAG GTGTGGTCAG GGACTATATA ATGGCTTGAT CCATGGTGGT GGGATGAGTA

84 1 TGGTTGGTTT AAGATCACCT GCTAACCACT TTTCATCAGA TGGGATTGGG AAGAGTAGTG 90 1 GAGATAGTTC CTCAGTTTCA CCCGTTCCTT ATGTGTTTAA TGGAAGTTTG AGGGGTAGGA

96 1 AAAGCAGTGC TGTGGAAAAG GTTGCTGAGA GGAGGCAGAG AAGAATGATA AAGAACCGAG

1021 AATCCGCTGC AAGATCACGA GCTCGCAAGC AGGCTTATAC AACGGAGTTG GAAGCAGAAG

10 81 TTGCTAAGCT GAAAGAGGAG AACCAAGAAT TGCAGAAGAA ACATGATCAC A CA AAAAA

1141 TACAGAAAAA TCAGGTGATG GAGATGATGA ATGTGCAGCC AGGAGCTAAG AAACGATGCT

12 01 TGCGACGAAC ACGGACAGGT CCTTGGTGAA C

According to SEQ ID NO:10 sequences Design pair of primers are as follows：

GhbZIP-3F: SEQ ID NO: 11：

ATGAACTTCAAGAACTTTGGA

GhbZIP-3R_:SEQ ID NO: 12：

TCACCAAGGACCTGTCCGT AP: SEQ ID NO: 13：

GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT passes through SEQ ID NO:11 and SEQ ID NO:12 clone GhbZIP-3 complete encoding sequences.

Cotton RNA is extracted, with primer SEQ ID NO:13 be reverse transcription primer, obtains cotton cDNA.Using Stratagene PfuUltra II Fusion HS DNA Polymerase, performing PCR reaction is entered by template of the cDNA of cotton.50 μ PCR reaction systems：The mM of 5 0.5 μ of μ lO PfuUltra II reaction Buffer 25 dNTP, 2.0 μ cDNA, the μ Μ of 1.0 μ PfuUltra II Fusion HS DNA Polymerase 10 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, 53 °C of annealing 30 s, 72 °C of 1 min of extension), 72 °C of 5 min of extension.

Pcr amplification product adds A tails：PCR primer adds the absolute ethyl alcohol of 2.5 times of volumes, and -20 °C of 10 min of placement, centrifugation is removed supernatant, dried, dissolved with 21 μ distilled waters.Add 2.5 μ Ι Ο χ Ε χ Buffer, 0.5 μ 5 mM dATP, 1.0 l Ex Taq.Reaction condition：70 °C of 30 min of reaction.Obtained about 1200bp DNA fragmentation is reclaimed（Omega QIAquick Gel Extraction Kits）, and be connected on pGEM T-easy carriers（Obtain

GhbZIP-3-pGEM plasmids）, then it is transformed into escherichia coli jm109 competent cell and is screened on the LB solid mediums containing 50 g/mL ampicillins（Method is ibid）.Random 8 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.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 gained sequence is SEQ ID NO:2, the amino acid sequence of its albumen encoded is SEQ ID NO: 1. ¥D¥¥OD¥ODO iiDOi¥OD¥¥ ¥0¥¥iDO¥00 ¥DDO¥DOiOi ¥¥0i¥0i¥0¥ OOi¥OiOO¥D T^TT i¥¥¥¥¥0¥D¥ i¥¥¥¥¥i¥Di ¥D¥Di¥Oi¥D ¥¥¥0¥¥0¥D0 ii¥¥0¥¥DD¥ ¥0¥00¥0¥¥¥ T80T

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OdOSi SOVO (the work OOHOdW aiV IZl aiOIS^a SI aHMAaaAi^H Sll^dl work 1S ￡ of 3 33 works of Η Λ Λ 0$ Η Λ of Ι Ξ Ο Ο Ο Λ a^OSdOl α 13 1) Ο Η Ο Ο Ο Ν Ο Λ OAOdSVWiOi 33$3 Μ Ι 19 The flat τ of ooooaosdd dNS Jsrai

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ZL 69linOZ OAV The GhbZIP-3 gene plant expression vector establishments of 12 01 CGGACAGGTC CTTGGTGA embodiments 3

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 Ν Ρ Τ Π genes contain double enhancers is replaced with Pnos promoters, to reduce expression of the Ρ Τ Π albumen in plant.35S promoter and Tnos terminator of the selection containing double enhancers are respectively as G) promoters of Z/P-3 genes and terminator, build flow as shown in Figure 1.

Use primer SEQ ID NO: M 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 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.Lj pCAMBIA2300 (Promega, T4 ligase box) are connected to as the PCR primer by obtained by after EcoRI, Bglll digestion and obtain pCAMBIA2300-l.

SEQ ID NO : 14 ：

GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO : 15：

ATCCAGATCTAGATCCGGTGCAGATTATTTG

Use 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：10 μ 5 xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ pBI121,1.0 μ Prime STAR, 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, (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.PCAMBIA2300-1 (Promega T4 ligases box) is connected to as the PCR primer by obtained by after Sall, EcoRI digestion and obtains pCAMBIA2300-2. SEQ ID NO : 16：

GC^rC^CGAATTTCCCCGATCGTTCAAA

SEQ ID NO : 17：

CAGAA rmXAGTGAATTCCCGATCTAGTA

Use primer SEQ ID NO:18 and SEQ ID NO:19 using pCAMBIA2300 plasmids as template amplification 35S promoters.Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems： 10 l 5 ><PS The mM of 3 μ of Buffer 2.5 dNTP, 1.0 μ dilute 50 times of pCAMBIA2300 plasmids, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:18 standing grain P SEQ ID NO:19 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, 50 °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: 18：

ACT^UrATGGTGGAGCACGACACTCT

SEQ ID NO: 19：

TGACf^ GAGATAGATTTGTAGAGAGAGAC

With SEQ ID NO:20 and SEQ ID NO:21 be primer, and the positive GMZH^-pGEM plasmids containing total length GhbZIP-3 genes is templates using in embodiment 2, and amplification two ends carry the GMZH^ genes of restriction enzyme site.Using stratagene PfuUltra II Fusion HS DNA Polymerase.50 μ PCR reaction systems：The mM of 5 0.5 μ of μ lO PfuUltra II reaction Buffer 125 dNTP, 2.0 μ GhbZIP-3-pGEM plasmids, the μ Μ of 1.0 μ PfuUltra II Fusion HS DNA Polymerase 10 primer SEQ ID NO:20 and SEQ ID NO: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, 58 °C of 30 s of annealing, s), 72 °C extend 5 min for 72 °C of extensions 45.Connected as the PCR primer by obtained by after Pstl, Sail digestion（Connection method is ibid）To pCAMBIA2300-3, plant expression vector 35S-GhbZIP-3-2300 (Fig. 2) is obtained.

SEQ ID NO: 20：

AAd ATGGGAGATAGATTCATGATG

SEQ ID NO: 21：

GC^G^CCATAAATGATTTCGCATACAC

35S-GMZ/ -3-2300 the expression vectors of embodiment 4 convert Agrobacterium

The preparation of Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competent cell：Agrobacterium LBA4404 is drawn to single spot inoculation on the LB solid mediums containing 50 g/ml rifampins and 50 g/ml streptomysins, 28 V 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_§Overnight incubation (about 12-16 h) is shaken in the LB fluid nutrient mediums of the streptomysins of/ι η 1, under 28 °C to OD_6(K)It is worth for 0.4, formation seed bacterium solution.Take and planted bacterium solution after 5 ml activation（1:20 ratio）100 ml are inoculated in containing 50 μ_§The rifampins of/η ι 1 and 50 μ_§In the LB fluid nutrient mediums of the streptomysins of/η ι 1,28 °C are shaken culture 2-2.5 h to OD_6QQ=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 certain Measure 4000 g under 10% glycerine resuspension thalline 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 35S-G of gained in 1 μ embodiments 3 added into the 40 μ competent cell) Zff-3-2300 plasmids, the min of ice bath about 10 after mixing.By the competent cell after ice bath and the positive 35S-G) mixtures of Z/P-3-2300 plasmids is transferred in the electric shock of ice precooling cup with micropipettor, and 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 of 1ml.It is quickly soft to be beaten mixture 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 transgenic arabidopsis using Agrobacterium-medialed transformation method

Plant culture to be transformed：By 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 incubators of 16h illumination/8h.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, incubator dark 16h illumination/8h and grown.The ml of 1/2MS fluid nutrient mediums 40 is poured before transplanting per alms bowl, backsight soil moisture is transplanted and keeps the skin wet in time.1/2MS fluid nutrient mediums are suitably poured in growth period.On demand per 3-4 weeks once（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, to release apical dominance, promotes the synchronous appearance of multiple secondary inflorescences.When the cm of most of inflorescences about 1 one 10 is high（Prepare to contaminate when cutting off after first inflorescence 4 one 8 d).

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）, 250 r/ min shakings incubated overnight under 28 °C of constant temperature.Resulting bacterium solution is inoculated into the 200 mL equally LB fluid nutrient mediums containing above-mentioned antibiotic in 1% -2% ratio again, 28 °C of constant temperature shakings make the concentration of Agrobacterium reach OD_6(K)=1.8, then 3000 r/min room temperatures, which centrifuge to use after 15 min, abandoning supernatant, contaminates culture medium（Silwet L-77 containing 5.0% sucrose and 0.05% (500 μ Ι Τ)) suspend Agrobacterium again, is suspended into OD₆₀₀About 0.80.

The dip-dye of inflorescence：The above-mentioned dip-dye culture medium containing Agrobacterium is added in big mouth container, each cm of bore 9 container Dip-dye culture medium containing Agrobacterium described in 200-300 mL of middle addition is 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.About 12-24 h remove covering after processing.Normal culture plant, the week of plant further growth 3-5, until silique browning is dried.T1 is harvested for transgenic seed, and by the seed centrifuge tube in 4 °C of lower dry storages.

Transgenic seed is screened:1/2MS fluid nutrient mediums are prepared, 0.8 % (W/V) agar powder is added, is heated to agar with micro-wave oven and dissolves completely, to be cooled to 50 °C or so, kanamycins are added to final concentration of 50 mg-L "¹, 25 mL are poured into every culture dish after shaking up, gained solid medium after experimental bench cooled and solidified is put and can be used to sowing.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, put after 4 °C of h of refrigerator cold treatment 72, move to and germinateed in the dark incubators of 23 V, 16h illumination/8h, 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.1/2MS fluid nutrient mediums are suitably poured in growth period.The g of kalamycin resistance Arabidopsis leaf 0.1 of growth 20 days is taken, DNA is extracted, with SEQ ID NO:11 and SEQ ID NO:12 amplification GhbZIP-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: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 30 s, 58 °C of annealing 30 s, 72 °C of 1 min of extension), 72 °C of 7 min of extension), the PCR plant for being accredited as the positive are numbered, plant is normally cultivated, the week of plant further growth 3-5, until silique browning is dried.T2 is harvested for transgenic seed（Numbering is：), T2F1-T2F16 and by seed centrifuge tube in 4 °C of lower dry storages.Embodiment 6 is overexpressed drought-enduring simulated experiments and Function Identification of the GhbZIP-3 transgenic arabidopsis T2 for plant and is impregnated with sterilized vermiculite with 1/2MS fluid nutrient mediums.Transgenic seed and control arabidopsis that numbering by the gained of embodiment 5 is T2F1-T2F8（Non-transgenic）Seed is sowed on the vermiculite respectively, 10 seeds are sowed per basin, 25 °C, the circulation of the h light cultures of 10 h optical cultures/14, pour a 1/2MS fluid nutrient medium within every 7 days, after culture 20 days, blade screening positive plant is applied using 2000 PPM kanamycins, per 4 more consistent kalamycin resistance seedlings of basin reservation size, for arid experiment.Transgenic arabidopsis and arid 14 days (not the watering) of control arabidopsis, 25 °C, 10 h optical cultures/14 h light cultures circulations.T2 shows that adjoining tree is all wilted seriously, and eight transgenic lines of T2F1, T2F2 T2F3 T2F4, T2F5, T2F6, T2F7, T2F8 totally 32 for the Identification of Drought of transfer-gen plant（Per each 4 of strain) in arabidopsis 30 can survive and continued growth shows significant drought tolerance（Referring to Fig. 3 a and 3b, with T2F1, Exemplified by T2F5, T2F2 T2F3 T2C 4 T2F6, T2F7 T2F8 result is similar with T2F1, T2F5, is not shown here）.Embodiment 7 verifies the expression of GhbZIP-3 genes on transcriptional level

Growth 20 days and then arid the control arabidopsis of 10 days after sprouting are taken respectively（Non-transgenic）Plant, significantly drought-enduring transgenic arabidopsis T2 are for plant（Point another ' J belongs to T2FK T2F2 T2F3 T2F4 T2F5 six strains of T2F6）Significantly drought-enduring transgenic arabidopsis T2 does not extract total serum IgE for each 0.05 g of blade of plant with plant RNA extracts kit (Invitrogen).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.Reverse transcription is carried out according to method shown in Invitrogen reverse transcription reagent box Superscript III Reverse Transcriptase（2 μ g total serum IgEs are used as template, reverse transcription primer SEQ ID NO： 13 ).Pass through SEQ ID NO：11 and SEQ ID NO:12 amplification GhbZIP-3, detect bZIP-3 albumen relative expression's situations.

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of above-mentioned reverse transcription.The Ρ Ο reaction systems of 50 μ 1： 10 μΐ 5 ><PS Buffer, the mM of 3 μ 2.5 dNTP, 2.0 μ cDNA, 1.0 l PrimeSTAR, 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 30 s of denaturation, s), 72 °C extend 10 min for 58 °C of annealing 30 s, 72 V extensions 45.

Product electrophoresis result is as shown in Figure 5：M is DNA Ladder Marker (DL2000, TakaRa), and 1-6 is notable drought-enduring transgenic arabidopsis T2 for plant（It is followed successively by： T2F1、 T2F2 T2F3 T2F4、 T2F5、 T2F6 )；7-10 is not significantly drought-enduring transgenic arabidopsis T2 for plant;11-14 is the control of non-transgenic arabidopsis.The electrophoretic bands of PCR products shown in figure size and it is in the same size（About 1200bp).As a result show, control arabidopsis does not have GhbZIP-3 transcriptions, and drought-enduring transgenic arabidopsis T2 is stronger for the transcription of GhbZIP-3 in plant, not drought-enduring transgenosis is intended ^ mustard T2 and do not transcribed for plant.

Claims

Claims

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

2. encoding the gene of the leucine zipper 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 35S-GMZ/P-3-2300 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 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.