CN102060919B - Three cotton ABF/AREB/ABI5/DPBF type transcription factors and coding genes and application thereof - Google Patents

Abstract

The invention discloses three novel cotton ABF/AREB/ABI5/DPBF type transcription factors GhABF2, GhABF3 and GhABI5, and coding genes and application thereof. Amino acid sequences of the GhABF2, GhABF3 and GhABI5 are shown as SEQ ID NO: 2, 5 and 8 respectively. Nucleotide sequences of the coding genes GhABF2, GhABF3 and GhABI5 are shown as SEQ ID NO: 1, 4 and 7 respectively. The invention also discloses the structural characteristics on genomic deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA) editing law of reading frame regions of the coding genes GhABF2, GhABF3 and GhABI5. The invention also discloses the cotton expression characteristics and yeast expression characteristics of the three genes and the characteristic that the three genes can improve the drought resistance of arabidopsis thaliana. The genes provide gene resources for culturing a plant variety resistant to abiotic stress, and have great significance for improving the abiotic stress resistance of plants.

Description

Three cotton ABF/AREB/ABI5/DPBF class transcription factors and encoding gene and application

Technical field

The present invention relates to three ABF/AREB/ABI5/DPBF class transcription factors that derive from cotton, name GhABF2, GhABF3, GhABI5 respectively, and their coding gene sequence.The invention still further relates to these three expression of gene spectrums, contain such expression carrier and utilize this genoid to cultivate the method for adversity resistant plant.

Background technology

Abiotic stress such as arid, high salt are the critical limitation factors of output of cotton and quality.In recent years, the aggravation that natural condition change is having a strong impact on the stable of output of cotton, has influenced the stable development of Cotton in China industry to a certain extent.Therefore, carry out the degeneration-resistant research of cotton, it is significant that the engineering means by plant gene improve its ability of restraining oneself to abiotic stresses such as arid, high salt.

ABA is the closest plant hormone of adverse circumstance signal conduction, under the abiotic stress conditions such as arid, high salt, and ABA synthetic and accumulation rapidly in the plant materials, along with alleviating that adverse circumstance stimulates, ABA promptly obtains decomposing (Taylor, I.B.Exp.Bot again, 2000,51:1563-1574); ABA is the important second messenger molecule of adverse circumstance signal conductive process, plant reply playing the part of in the adverse circumstance signal conductive process important role (Christmann A.Plant Biol, 2006,8:314-325); ABA is one of main working substance of cross-adaptation, (ShinozakiK such as Shinozaki, J Exp Bot, 2007,58 (2): 221-227) Arabidopis thaliana environment stress expression of gene study on regulation is found that the gene of the gene of environment stress abduction delivering and ABA abduction delivering exists important interaction.Exist between environment stress start signal and the genetic expression and rely on ABA and do not rely on two kinds of approach of ABA (Nakashima K, Plant Physiol, 2009,149 (1): 88-95).There is a kind of path that does not need protein synthesis in the adverse circumstance signal transduction path of dependence ABA, rising by ABA content in the inducing plant cell under the environment stress activates the adjusting albumin A BF/AREB/ABI5/DPBF that a class has alkaline leucine zipper structure, such transcription factor by with ABRE motif (pyACGTGGC, ABAresponsive element) specific combination, the expression of regulation and control drought resisting functional gene.

ABF/AREB/ABI5/DPBF class transcription factor belongs to the bZIP proteinoid, such transcription factor N end comprises three conservative structural domains, be transcriptional activation domain, the C end comprises a conservative alkaline leucine zipper structure, for transcribing binding domains (Choi H, PlantPhysiol, 2005,139:1750-1761).So far, 9 ABF/AREB/ABI5/DPBF homolog: AREB1/ABF2 have been had been found that in the arabidopsis thaliana chromosome genome, AREB2/ABF4, AREB3/DPBF3, ABF1, ABF3/DPBF5, ABI5/DPBF1, EEL/DPBF4, DPBF2 and AT5G42910 (Suzuki, M.PlantPhysio1.2003,132:1664-1677).Arabidopis thaliana ABF1, AREB1/ABF2, ABF3/DPBF5, AREB2/ABF4, ABI5/DPBF1 etc. are discovered, they have mainly participated in environment stress responsing reactions such as ABA, arid, salt marsh, low temperature, (Takashi F plays an important role in the adverse circumstance signal conduction that relies on ABA, Pro Natl Acad Sci USA, 2006,103 (6): 1988-1993).The ABRE motif is all contained in the promoter region of many ABA abduction delivering genes, therefore, by importing or improve this adverse circumstance correlated transcription factor gene and can regulate and control a series of adverse circumstance Expression of Related Genes, thereby improves the comprehensive anti-adversity ability of plant.Yet have not yet to see the report of ABF/AREB/ABI5/DPBF genoid in important cash crop cotton.

Summary of the invention

An object of the present invention is to provide three ABF/AREB/ABI5/DPBF class transcription factors in the cotton, difference called after GhABF2, GhABF3, GhABI5, they are respectively the protein with SEQ ID NO:2 in the sequence table, amino acid residue sequence of 5,8, or with SEQ ID NO:2,5,8 amino-acid residue through replacement, disappearance or the interpolation of one or several amino-acid residue have with sequence SEQ IDNO:2,5,8 the identical activity of amino acid residue sequence by sequence SEQ ID NO:2,5,8 protein of deriving.

GhABF2 preferably has the protein of sequence SEQ ID NO:2 amino acid residue sequence in the sequence table, is made up of 417 amino-acid residues; GhABF3 preferably has the protein of sequence SEQ ID NO:5 amino acid residue sequence in the sequence table, is made up of 407 amino-acid residues; GhABI5 preferably has the protein of sequence SEQ ID NO:8 amino acid residue sequence in the sequence table, is made up of 422 amino-acid residues.

Another object of the present invention provides three cotton ABF/AREB/ABI5/DPBF class transcription factor GhABF2, encoding gene GhABF2, the GhABF3 of GhABF3, GhABI5, GhABI5, be respectively with sequence list in SEQ ID NO:1,4,7, limit dna sequence dna and have 90% above similarity, and coding identical function protein DNA sequence.

GhABF2 is the dna sequence dna of SEQ ID NO:1 in the sequence list preferably, and by the 2027bp based composition, the reading frame of this gene is the dna sequence dna from the 1251bp based composition of the 442nd to 1692 at 5 ' end; GhABF3 is the dna sequence dna of SEQ ID NO:3 in the sequence list preferably, and by the 1963bp based composition, the reading frame of this gene is the dna sequence dna from the 1221bp based composition of the 333rd to 1553 at 5 ' end; GhABI5 is the dna sequence dna of SEQ ID NO:7 in the sequence list preferably, and by the 1927bp based composition, the reading frame of this gene is the dna sequence dna from the 1266bp based composition of the 384th to 1649 at 5 ' end.

A further object of the present invention provides the opening of three cotton ABF/AREB/ABI5/DPBF class transcription factor GhABF2, GhABF3, GhABI5 encoding gene GhABF2, GhABF3, GhABI5 and reads the code structure genome nucleotide sequence, be respectively SEQ ID NO:3,6,9 in the sequence list, limit exon (Exon) dna sequence dna and have 90% above similarity, intron (Intron) DNA has 80% above similarity.

GhABF2 is the dna sequence dna of SEQ ID NO:3 in the sequence list preferably, by the 2177bp based composition, comprise 4 Exon and 3Intron, Exonl (1～1077) comprises the dna sequence dna of 1077bp base, the dna sequence dna that Exon2 (1620～1691) comprises the 72bp base, the dna sequence dna that Exon3 (1788～1817) comprises the 30bp base, the dna sequence dna that Exon4 (2106～2177) comprises the 72bp base, reads code structure by the final opening that forms the 1251bp base sequence of RNA montage processing.

GhABF3 is the dna sequence dna of SEQ ID NO:6 in the sequence list preferably, by the 1773bp based composition, comprise 4 Exon and 3Intron, Exonl (1～1047) comprises the dna sequence dna of 1047bp base, the dna sequence dna that Exon2 (1321～1392) comprises the 72bp base, the dna sequence dna that Exon3 (1485～1514) comprises the 30bp base, the dna sequence dna that Exon4 (1705～1776) comprises the 72bp base, reads code structure by the final opening that forms the 1221bp base sequence of RNA montage processing.

GhABI5 is the dna sequence dna of SEQ ID NO:9 in the sequence list preferably, by the 1767bp based composition, comprise 4 Exon and 3Intron, Exonl (1～1071) comprises the dna sequence dna of 1071bp base, the dna sequence dna that Exon2 (1235～1306) comprises the 72bp base, the dna sequence dna that Exon3 (1413～1442) comprises the 30bp base, the dna sequence dna that Exon4 (1675～1767) comprises the 72bp base, reads code structure by the final opening that forms the 1266bp base sequence of RNA montage processing.

A further object of the present invention provides expression vector (Fig. 4) pBI121-GhABF2, pMD18-GhABF3, the pMD18-GhABI5 (depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center of containing mentioned GhABF2, GhABF3, GhABI5 class transcription factor; Address: No. 1 institute in Beichen Lu, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Deposit number: CGMCC No.3152, CGMCC No 3153, CGMCC No 3154; Preservation date: on 06 30th, 2009; Classification name: colon bacillus Escherichia coli).With any expression vector that can guide foreign gene in plant, to express.Contain this invention GhABF2, GhABF3, GhABI5 expression carrier and clone, and the plant lines kind that contains this genoid is protection scope of the present invention.

Expression vector of the present invention can infect by using Agrobacterium, pollen tube channel, particle gun conversions etc. import vegetable cell, can use method plant transformed host of the present invention to comprise monocotyledons and dicotyledons, for example: cotton, paddy rice, wheat, corn, turfgrass, willow, cucumber, clover etc.

Description of drawings

Fig. 1 represents that it is template pcr amplification result's agarose electrophoresis figure with genomic dna and eDNA respectively that code structure is read in GhABF2, GhABF3, the opening of GhABI5 encoding gene.

Fig. 2 is GhABF2, GhABF3, GhABI5 gene structure synoptic diagram

Fig. 3 is PCR result's electrophoretogram, shows GhABF2, GhABF3, the expression of GhABI5 in cotton different tissues organ.

Fig. 4 is the polyacrylamide gel electrophoresis collection of illustrative plates, shows the molecular weight of GhABF2 yeast expression.

Fig. 5 is yeast growth figure, and showing changes GhABF2 gene yeast OD under different PEG6000 and NaCl concentration ₆₀₀Curve.

Fig. 6 is the structure signal physical map of GhABF2 transgene expression vector.

Fig. 7 is for changeing the drought resisting experimental result picture of GhABF2, GhABF3, GhABI5 gene Arabidopis thaliana.

Embodiment

Clone's materials and methods of 1 three cotton ABF/AREB/ABI5/DPBF of embodiment class transcription factor GhABF2, GhABF3, GhABI5 encoding gene

1) cotton material: cotton material is selected free kind Y18R for use.

2) bacterial classification: intestinal bacteria E.coliDH5 α, Agrobacterium LBA4404 and pichia spp GS115.

3) carrier: pMD18-T, pG4AB, pBI121, pJawohl8-RNAi and pPIC9.0K.

4) toolenzyme and modifying enzyme: various restriction enzymes and modifying enzyme are available from TaKaRa company, NEB company and Fermentas company.

5) chemical reagent: pharmaceutical chemicals is domestic and international analytical pure.

6) primer is synthetic: synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

7) order-checking: finished by the big genome company of China.

Experimental procedure

1) preparation of total RNA: adopt the hot borate method of improvement to extract total RNA at face kind Y18R blade position, land, be used for RT-PCR, Tail-PCR, 3 ' RACE etc.

2) obtain the conservative segment est sequence of ABF/AREB/ABI5/DPBF class transcription factor gene in conjunction with information biology: the ABF/AREB/ABI5/DPBF class transcription factor constructional feature of including according to GenBank storehouse and patent database, in conjunction with 6 degenerated primers of conserved domain sequences Design:

FP1：5′-TGGARGAGTTTYTRGTYAGAGC-3′；

FP2：5′-TKGAGGATTTCTTGGTKAARGC-3′；

RP1：5′-GCYTGTTTTCTKGCTCTAGATC-3′；

RP2：5′-GCCTGCTTSCGDGCCCKTGAYC-3′；

bZIPFP1：5′-AWGATHAARAAYMGVGARTCHGCDGCDMGDTCHMGVGC-3′；

bZIPFP1：5′-GARTCHGCDGCDMGDTCHMGVGCHMGVARRCARGCWYAH-3′；

(M：AorC；K：GorT；W：AorT；R：AorG；Y：CorT；S：CorG；D：AorGorT；H：AorCorT；N：AorCorGorT)

FP1, FP2 and RP1, RP2 make up amplification in twos and obtain two purpose amplifications, and size is respectively 660bp, 590bp; BZIPFP1 and bZIPFP1 and 3 ' RACE combination of primers nest-type PRC get a purpose amplification, and size is 553bp; Reclaim respectively, is connected with the pMD18-T carrier, 16 ℃ spend the night, transformation receptor bacterium DH5 α, bacterium liquid PCR and enzyme cut and identify that correct bacterial strain send China big gene sequencing, according to analytical results difference called after GhABF2, GhABF3, the GhABI5 of BLAST.

3) acquisition of GhABF2, GhABF3, GhABI5 full length cDNA sequence:

Adopt the method for Tail-PCR and 3 ' RACE to obtain GhABF2, GhABF3, GhABI5 full length cDNA sequence, Tail-PCR presses the Genome Walking Kit of TaKaRa company operation, 3 ' RACE press TaKaRa company 3 '-Full RACE Core Set Ver.2.0 operates.

Be used for Tail-PCR with power traction: AD1:5 '-NTCGASTWTSGWGTT-3 '

AD2：5′-NGTCGASWGANAWGAA-3′

AD3：5′-TCTTICGNACITNGGA-3′

AD4：5′-TTGIAGNACIANAGG-3′

(S：CorG；W：AorT；N：AorCorGorT)

The special primer that is used for TAIL-PCR: GhABF2sp1:5 '-CCTCCATTTCCCACTAGGCCAACAC-3 '

GhABF2sp2：5′-CTTTGCCTATTCCACCCATTGTGC-3′

GhABF2sp3：5′-GGTTTATTACCACCGCCGCCATCAC-3′

GhABF3sp1：5′-CCCACCACTCTGCAGGCCACTAGACTG-3′

GhABF3sp2：5′-GTGGCTGTTGTTTCTGCTGTGAATGTGATG-3′

GhABF3sp3：5′-CACCAACCGGTTGCATATCTTCCCTCAC-3′

GhABI5sp1：5′-GCCATCGACCGTCATTGTAGAGCTC-3′

GhABI5sp2：5′-TCGACGACGCCATATTCGGCGGAG-3′

GhABI5sp3：5′-TCAGTCTTCCCCGAGATGTTGAGC-3′

Be used for 3 ' RACE primer:

AMVOligo：5′-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTT-3′

RACErv1：5′-GCTGTCAACGATACGCTACGTAACG-3′

RACErv2：5′-CGCTACGTAACGGCATGACAGTG-3′

The special primer that is used for 3 ' RACE:

GhABF2fp1：5′-AGCACCAGTTGGCTCAGCAG-3′

GhABF3fp 1：5′-AGAGAGTCAGCTGCAAGATC-3′

GhABI5fp1：5′-GATAGAGATAGGTGGACTAC-3′

The dna fragmentation that amplifies is separated, reclaims and be connected to pMD18-T easy Vector with 1% agarose gel, Transformed E .coli DH5 α, after cutting and identify correctly, enzyme send China big gene sequencing, utilize biosoftware Vector9.0, Conting that the sequence that obtains is spliced, obtain the full length cDNA sequence of GhABF2, GhABF3, GhABI5 gene, and verify according to the sequences Design primer that obtains, 3 ' end primer is RACE rv1 and RACE rv2,5 ' end checking primer: GhABF2fp2:5 '-ATTTCATTTAGAAAACTGGGT-3 '

GhABF3fp2：5′-GTTGTTTCTGCTGTGAATGTG-3′

GhABI5fp2：5′-AGTGAGAAGAACAAAAGAGGC-3′

Obtain the full length cDNA sequence of GhABF2, GhABF3, GhABI5 gene at last, be respectively SEQ ID NO:1,4,7 in the sequence table, infer that according to the eDNA sequence its protein sequence is SEQ ID NO:2,5,8 in the sequence list.

Embodiment 2 GhABF2, GhABF3, GhABI5 transcription factor gene constructional feature are analyzed

According to the full length cDNA sequence of acquisition GHABF2, GHABF3, GHABI5 gene, in conjunction with rotaring intertranslating start site and the termination site design primer of information biology prediction, do template with cotton genomic dna and cDNA respectively and carry out pcr amplification.

The primer that is used for PCR: GhABF2fp3:5 '-ATGGGGACTAACATGAACTT-3 '

GhABF2rv3：5′-CCAAGGACCGGTCTGGGTTC-3′

GhABF3fp3：5′-ATGGGGTCTAATCTGAATTT-3′

GhABF3rv3：5′-CCAAGGGCCTGTCAGTGTTC-3′

GhABI5fp3：5′-ATGGTGGTTGAGAACTCTGA-3′

GhABI5rv3：5′-TAATGGACCACTTAGATTTC-3′

The segment that will increase reclaims, is connected to pMD18-T easy Vector, Transformed E .coli DH5 α, after cutting and identify correctly, enzyme delivers to the big gene sequencing of China, cDNA sequence and the genomic dna sequence of code structure read in the opening that obtains GhABF2, GhABF3, GhABI5 gene at last, is respectively the sequence SED ID NO:3,6,9 in the sequence table.CDNA and genomic dna sequence comparison are found, the opening of GhABF2, GhABF3, GhABI5 gene is read code structure and all is made up of 4 Exon and 3 Intron on genome, and RNA shearing processing meets the rule of " GT ... AG " (among Fig. 1 fully, M:Marker, CK-: negative control, A1, B2, C3 are respectively that GhABF2, GhABF3, GhABI5 are template pcr amplification result with the cotton genomic dna, and D4, E5, F6 are respectively that GhABF2, GhABF3, GhABI5 are template pcr amplification result with cotton eDNA; Among Fig. 2, GhABF2, GhABF3, three gene structure synoptic diagram of GhABI5)

The expression characterization analysis in cotton of embodiment 3 GhABF2, GhABF3, GhABI5 transcription factor gene

1) template preparation: the hot borate method that adopts improvement is from cotton Different Organs material: extract total RNA root, stem, leaf, calyx, petal, stamen, ovary, fiber, bud, the cotton boll, the RNA quality of extraction is by OD ₂₆₀/ OD ₂₈₀Ratio and 0.7% agarose gel electrophoresis are identified.Be that template is carried out reverse transcription reaction by following scheme with it: add the total RNA of 2 μ g and 1 μ L Oligo (dT) in a PCR tubule, 65 ℃ of incubation 15min add 5 * MMLV (RNase H then on ice ^-) Buffter 5 μ L, dNTP (25mmol/L) 5 μ L, Ribonuclease Inhibitor 20U, MMLV 200U handles water with DEPC at last and is supplemented to cumulative volume 25 μ L, 42 ℃ of incubation 1h, 95 ℃ of water-bath 5min deactivation MMLV, the cDNA-20 that obtains ℃ of preservation is standby.

2) design of the relative quantification of template cDNA and interior label primer: cotton actin gene (Cotton Actinlgene:Accession NO.AY305723) the confidential reference items design of primers that goes up login according to GenBank is as follows:

GhActfp：5′-CTCTCTCTGTATGCCAGTGGTC-3′

GhActrv：5′-TTGTCCGTCAGGCAACTCATAG-3′

CDNA does template with cotton, carries out the band that pcr amplification will obtain 321bp with this primer.

PCR reaction system: template 1 μ l, PCR Buffer 5 μ l, 10mMdNTP 3 μ l, GhActfp 1 μ l, GhActrv 1 μ l, Taq 1U, ddH ₂O8 μ l.

The PCR condition: 94 ℃, 2min; 94 ℃, 30Sec; 55 ℃, 30Sec; 72 ℃, 30Sec; 30cycle; 72 ℃, 10min.

Electrophoresis result according to the PCR product is diluted template cDNA, adjusts the consumption of template cDNA, and the amount basically identical of the DNA band that goes out up to GhActfp and GhActrv primer amplification makes the content basically identical of template cDNA in every microlitre solution.

4) pcr amplification of GhABF2, GhABF3, GhABI5 gene:

1. GhABF2, the primer that relates to pcr amplification is as follows:

GhABF2fp4：5′-TCCAAATTTGATGGGAACTAG-3′

GhABF2rv4：5′-CTGCTTCCAATTCCATTGTAT-3′

Carry out pcr amplification with this primer, if cDNA does can the increase band of 545bp of template, if genomic dna is done can the increase band (the 542bp intron that contains) of 1087bp of template.

PCR system: template 1 μ l, PCR Buffer 5 μ l, 10mMdNTP 3 μ l, GhABF2fp4 1 μ l, GhABF2rv4 1 μ l, Taq 1U, ddH ₂O 8 μ l.

The PCR condition: 94 ℃, 2min; 94 ℃, 30Sec; 55 ℃, 30Sec; 72 ℃, 30Sec; 30cycle; 72 ℃, 10min.

2. GhABF3, the primer that relates to pcr amplification is as follows:

GhABF3fp4：5′-TTTGAGTAACAATAACTCAGT-3′

GhABF3rv4：5′-TTTGCAACTTCAGCTTCAAGT-3′

Increase with this primer, if cDNA does can the increase band of 494bp of template, if genomic dna is done can the increase band (the 273bp intron that contains) of 774bp of template.

PCR system: template 1 μ l, PCR Buffer 5 μ l, 10mMdNTP 3 μ l, GhABF3fp4 1 μ l, GhABF3rv4 1 μ l, Taq1U, ddH ₂O 8 μ l.

The PCR condition: 94 ℃, 2min; 94 ℃, 30Sec; 55 ℃, 30Sec; 72 ℃, 30Sec; 30cycle; 72 ℃, 10min.

3. GhABI5, the primer that relates to pcr amplification following (amplification 564bp, 727bp)

GhABI5fp4：5′-CATTCACAACATCAACAACAG-3′

GhABI5rv4：5′-CTGCTTGAGGTGAGTATTCTC-3′

Increase with this primer, if cDNA does can the increase band of 564bp of template, if genomic dna is done can the increase band (the 163bp intron that contains) of 727bp of template.

PCR system: template 1 μ l, PCR Buffer 5 μ l, 10mMdNTP 3 μ l, GhABI5fp4 1 μ l, GhABI5rv4 1 μ l, Taq1U, ddH ₂O 8 μ l.

The PCR condition: 94 ℃, 2min; 94 ℃, 30Sec; 55 ℃, 30Sec; 72 ℃, 30Sec; 30cycle; 72 ℃, 10min.

Electrophoresis result shows: GhABF2 has expression in Radix Gossypii, stem, leaf, bud, cotton boll, expression amount in the root, stem and leaf is high slightly, and along with the growth GhABF2 expression amount of bud constantly raise (among Fig. 3, A:CK+, B:CK-, C: root, D: stem, E: leaf, F: calyx, G: petal, H: stamen, I: ovary, J: fiber, K: bud, L: cotton boll; M:CK+, N:CK-, O: cotton boll 1, P: cotton boll 2, Q: bud 1, R: bud 2, S: bud 3, T: bud 4); In root, stem, leaf, calyx and the bud of GhABF3 in cotton expression is arranged, the expression amount in the root, stem and leaf is high slightly; In the root of GhABI5 in cotton, stem, leaf flower, the flower bud expression is arranged, the expression amount in the cotton boll is high slightly.

Embodiment 4 GhABF2 transcription factors expression analysis in pichia spp

1) pichia spp GS115 competent cell preparation: picking pichia spp recipient bacterium GS115 (His-, Mut+) single colony inoculation is in the 10mLYPD liquid nutrient medium, 30 ℃ of shaking table overnight incubation, transfer in 100mL YPD substratum with 1% inoculum size, 30 ℃ are cultured to OD600=1.3～1.5 again; 4 ℃ of centrifugal 5min of 5000rpm, supernatant discarded, sterilized water with the precooling of 100mL ice is resuspended with thalline, 4 ℃ of centrifugal 10min of 5000rpm, supernatant discarded, with the sterilized water of 50mL ice precooling that thalline is resuspended, 4 ℃ of centrifugal 10min of 5000rpm, with 20mL 1mol/L sorbyl alcohol washing 1 time, be dissolved in the sorbyl alcohol of 200 μ L 1mol/L ice precooling, in order to transforming again.

2) Yeast expression carrier makes up: code structure is read in the GhABF2 gene opening that obtains in the pcr amplification process be connected on the pUC19 carrier, be connected with the pPIC9.0K carrier that EcoR I enzyme is cut processing with SnaB I behind recycling SnaB I and the EcoR I double digestion, be built into external secretion type Yeast expression carrier pPIC9.0KGhABF2.

3) primary dcreening operation of the conversion of the electric shock of yeast and recon: utilize restriction endonuclease Sal I linearizing to express Yeast expression carrier pPIC9.0KGhABF2, transfer to after getting 80 μ L competent cells and linearization plasmid (1～5 μ g) mixing in the 0.2cm electric shock cup of ice precooling, electric shock transformed yeast competent cell (2.5KV, 5ms), in the electric shock cup, add the ice-cold 1mo1/L sorbyl alcohol of 1mL immediately, behind the mixing, be applied on the RDB flat board with every plate 200 μ L bacterium liquid, 30 ℃ are cultured to and grow transformant.

4) yeast recon methyl alcohol utilizes determining of type and PCR to detect: with the dull and stereotyped and MD flat board of recon difference dibbling MM that aseptic toothpick picking screens above, dibbling simultaneously is according to bacterial strain GS115 Albumin (His4 ⁺Mut ^S) and GS115 β-Gal (His4 ⁺Mut ⁺).Consistent with the MD growth at MM, its phenotype is Mut ⁺Normal in MD growth, poor growth or long on MM, its phenotype is Mut ^SThe recon bacterium colony of picking fresh culture is suspended in the 10ul sterilized water, adds 5u15U/ul yeast lyase, 30 ℃ of incubation 10min, and liquid nitrogen freezing 1min then is as template.Used amplimer is:

5′AOX I：5′-CGACTGGTTCCAATTGACAAGC-3′

3′AOX I：5′-GGCAAATGGCATTCTGACATCC-3′

PCR system: template 1 μ l, PCR Buffer 5 μ l, 10mMdNTP 3 μ l, 5 ' AOX I, 1 μ l, 3 ' AOX I, 1 μ l, Taq 1U, ddH ₂O8 μ l.

The PCR condition: 94 ℃, 2min; 94 ℃, 30Sec; 55 ℃, 30Sec; 72 ℃, 30Sec; 30cycle; 72 ℃, 10min.

5) sds polyacrylamide gel electrophoresis of protein (SDS-PAGE): the separation gel solution (H2O1.6ml of sds page preparation 12%, 30% acrylamide mother liquor 2.0ml, 1.5mol/LTris-Cl (pH8.8) 1.3ml, 10%SDS 0.05ml, 10% ammonium persulphate 0.05ml, TEMED 0.002ml) and concentrated sol solution (H2O1.4ml, 30% acrylamide mother liquor 0.33ml, 1.0mol/L Tris-Cl (pH6.8) 0.25ml, 10%SDS 0.02ml, 10% ammonium persulphate 0.02ml, TEMED 0.002ml), concentrate glue in the separation gel perfusion, insert comb, polymerized at room temperature 45 minutes.The processing of sample: get the 1ml nutrient solution in the centrifugal collection thalline of 1.5ml Eppendorf tube, 500 μ l TE (pH8.0) wash thalline one time, add 20 μ l H2O and 20 μ l, 2 * sample buffer suspension thalline, 100 ℃ were boiled 3 minutes, remove cell debris in centrifugal 10 minutes in 4 ℃ with 12000rpm, get sample electrophoresis on the 2 μ l supernatants.Electrophoresis: 8v/cm electrophoresis to dyestuff forward position enters separation gel, and voltage is increased to 15v/cm, continues electrophoresis to dyestuff and arrives separation gel bottom, following glue.Dyeing, decolouring: the staining fluid that adds at least 5 times of volumes soaks gel, is placed on the shaking table that shakes gently room temperature dyeing more than 4 hours.The SDS-PAGE electrophoresis result shows, GhABF2 albumen size is about 44KD (among Fig. 4, M:Marker, A: negative control, B:GhABF2 albumen, size is 44KD).

6) influence of the pichia spp of different concns PEG600, NaCl growth: with GhABF2 gene recombination bacterium GS115/GhABF2 and control strain GS115/pPIC9K inducing culture under the liquid nutrient medium condition shown in the table 1 respectively, picking mono-clonal bacterial strain was cultivated 48 hours in 28 ℃ of 250rpm/min of BMGY substratum, shaking bottled liquid measure is 10ml/100ml, changing 28-30 ℃ of 250rpm/min of BMMY substratum then cultivates, mended starting point concentration methyl alcohol in per 24 hours, and cultivated and detect cell density OD after 48 hours ₆₀₀According to the cell density OD that records ₆₀₀Data are drawn under the different oxygen supply conditions growth curve of reorganization bacterium GS115/9GhABF2 and control strain GS115/pPIC9K respectively.

PEG6000, NaCl concentration arrange in the table 1 BMMY substratum

The result shows: PEG6000 and concentration are the adverse circumstance environment of yeast growth breeding greater than 2.0%NaCl, influences the growth of yeast strain, and along with the rising of concentration, the yeast strain and the unloaded bacterial strain of commentaries on classics that change the GhABF2 gene all detect OD ₆₀₀Value descends, and illustrates that growth and breeding speed slows down.In PEG6000 concentration between 0～4.0% o'clock, along with the rising of PEG6000 concentration, the control strain OD that changes the GS115/pPIC9KGhABF2 yeast strain and change GS115/pPIC9K for 1, No. 2 ₆₀₀Value all descends, and changes unloaded yeast strain OD ₆₀₀Value descends faster, reaches at 1.33% o'clock in PEG6000 concentration, changes the OD of GhABF2 gene yeast bacterial strain ₆₀₀Be respectively 1.381,1.307, change unloaded yeast strain OD ₆₀₀Value is 1.095, changes goal gene yeast strain 1 and 2 than changeing unloaded yeast strain OD ₆₀₀Value respectively high by 26.12%, 19.36%, the difference of the two reaches maximum value, the continuation along with PEG6000 concentration then raises the two OD ₆₀₀Value is gradually consistent.NaCl concentration is similar with PEG6000 for the influence of yeast strain growth between 1.66～5.0% o'clock, along with the rising of NaCl concentration, and the control strain OD that changes the GS115/pPIC9KGhABF2 yeast strain and change GS115/pPIC9K for 1, No. 2 ₆₀₀Value all descends, and changes unloaded yeast strain OD ₆₀₀Value descends faster, when NaCl concentration is 3.66%, changes the yeast strain 1 of GhABF2 gene and 2 OD ₆₀₀Value is respectively 0.367,0.349, changes unloaded yeast strain OD ₆₀₀Value is 0.212, changes goal gene yeast strain 1 and 2 than changeing unloaded yeast strain 0D ₆₀₀Value respectively high by 73.11%, 64.62%, the difference of the two reaches maximum value, the continuation along with PEG6000 concentration then raises the two OD ₆₀₀Value is gradually consistent.Above result can illustrate, PEG6000 and concentration are the adverse circumstance environment of yeast growth breeding greater than 2.0%NaCl, (among Fig. 5, A: yeast growth relatively under the different concns PEG6000 and the yeast strain that changes the GhABF2 gene can be improved the ability of restraining oneself to PEG6000, NaCl adverse circumstance environment to a certain extent; B: yeast growth relatively under the different concns NaCl).

The structure of embodiment 5 GhABF2, GhABF3, GhABI5 transgene expression vector

1) GhABF2 gene plant expression vector establishment: GhABF2, GhABF3, GhABI5 be building up to obtain pBI121GhABF2 (Fig. 6), pBI121GhABF3, pBI121GhABI5 on the plant expression vector pBI121, transform DH5 α, extract plasmid, enzyme is cut evaluation, choose needed clone, send China big gene sequencing, and it is transformed Agrobacterium LBA4404.

2) Arabidopis thaliana of GhABF2, GhABF3, GhABI5 gene transforms:

Agrobacterium is cultivated: transfer and identify the single colony inoculation of correct Agrobacterium at 3ml YEB (Kan50mg/L, Streptomycin sulphate 50mg/L), 28 ℃, 250rpm cultivates 36h; Transfer among the fresh YEB of 200ml (Kan50mg/L, Streptomycin sulphate 50mg/L) by 1:500,28 ℃, 250rpm cultivates 24h, to OD ₆₀₀≈ 1.0; 5000rpm, 4 ℃, the centrifugal collection thalline of 10min; Resuspended thalline is in penetrating fluid (1/2MS+5% sucrose, pH6.0,120 ℃, the sterilization 15min of 1.5 times of volumes; Be 0.044uM with adding 6-BA before to final concentration, the VB6 final concentration is 1mg/L, and Silwet is 0.02%.

The cultivation of Arabidopis thaliana: 4 ℃ of spring flowers of Arabidopis thaliana seed were handled 2-3 days, 4-5 seed (nutrition soil: vermiculite=2: 1) of every basin sowing; In the greenhouse, cultivate (22 ℃, illumination 16h); Treating that Arabidopis thaliana is extracted out just behind the mossy cuts off it, when treating that it extracts enough inferior mossies out, can be used for conversion.

Arabidopis thaliana transforms: the bud of Arabidopis thaliana is immersed in the penetrating fluid, vacuumize 1min; After conversion finishes, secretly cultivate 24-48h, can normally cultivate then.

Seed collection and screening: weighing 20g Arabidopis thaliana seed is in the 1.5mL centrifuge tube, the ethanol (containing 0.05%Tween20) that adds 1mL75%, shook 10 minutes, the centrifugal supernatant that goes, washing with alcohol 2-3 time that adds 1mL95%, the ethanol that adds 0.5mL 100% in super clean bench is transferred on the aseptic filter paper, dries up; Seed broadcasting after drying up is to 1/2MS flat board (Kan50mg/L); 4 ℃, after 2 days, 22 ℃, the 16h illumination cultivation; Positive plant is transplanted in the nutrition soil cultivates, collect seed and carry out T1 for screening.

Transgenic arabidopsis is identified: the genomic dna that extracts positive Arabidopis thaliana plant is used for PCR and detects.

Forward primer: 35S FP:5 '-CACAATCCCACTATCCTTCG-3 '

Reverse primer: GhABF2 rv5:5 '-CTTCCCAATCCCATCTGACG-3 '

GhABF3rv5：5’-CCCACCACTCTGCAGGCCAC-3’

GhABI5rv5：5’-GCCATCGACCGTCATTGTAG-3’

PCR system (50ul): template (transgenic arabidopsis DNA) 1 μ l, PCR Buffer 5 μ l, 10mMdNTP 3 μ l, each 1ul of primer; Add sterilized water to 50ul.

The PCR condition: 94 ℃, 2min; 94 ℃, 30Sec; 55 ℃, 30Sec; 72 ℃, 30Sec; 30 cycle; 72 ℃, 10min.

Reaction system: 1ul (30ng); 10 * Buffer 2ul; Taq enzyme (0.5ul); DNTP (3ul);

Embodiment 6 GhABF2, GhABF3, the analytical test of GhABI5 transfer-gen plant drought resistance

Transfer-gen plant and non-transgenic Arabidopis thaliana plant are placed the cultured continuously 12 days of not feeding water under the normal growth conditions, and the blade of transgenic arabidopsis plant is still acted normally as a result, and the wild-type Arabidopsis leaf is all flavescence then, even withered; The survival of back transgenic arabidopsis 100% of one week of rehydration is got off, non-transgenic plant survival rate less than 50%, and the serious hysteresis of growing appears.Above result shows that GhABF2, GhABF3, GhABI5 can obviously improve the drought resisting performance of Arabidopis thaliana plant, and result such as Fig. 6, A are the plant that arid was handled 12 days, and B is the plant of rehydration after one week.

Industrial or agricultural is used

The clone of success of the present invention 3 cotton ABF/AREB/ABI5/DPBF class transcription factor GhABF2, GhABF3, GhABI5 encoding gene, and successfully it is imported in yeast and the Arabidopis thaliana, obtained drought-resistant transgenic yeast and the transgenic arabidopsis that waits abiotic stress, provide GENE SOURCES for cultivating the drough-resistant and saline-alkali resistant new variety of plant, had important theory and realistic meaning to cultivating resisting abiotic adversity plant new variety.

Sequence table

＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences

<160>9

<210>1

<211>2027

<212>DNA

<213>Gossypium hirsutum Y18R

<220>

<221>CDS

<222>(442)..(1692)

<400>1

ATTTCATTTAGAAAACTGGGTTTTCTTTTGAATAATTAATCGACTCAGATGTAGTATTGGATTTTTTTAATGTTTCGGGGGCGGG 85

ATCTGATGTTTTAATTTTTCAAGGGTTTGGCTTGTTAAATTTCTTGAGCACATTGGAATGTTAGGTGTTACTAGCACTTGCAGAA 170

CAAAAAAAAGAAAGAGTTGGTCTTTACTTCCCCTTTTGTGTTTATGTTTTTTTATTGTTTTTCTTAGTTAAAGATTTCCTGTATA 255

GATCTTGATTTTTGGGTTTTTCCTTTTTTTTTTAACGTGTACTTCCCAAATATAGAAATATGTAAATCATTCAAACTGCATGGGA 340

TTTATTTTATCTGTTTTATTTTCATTGAAGTAGTAGTTAAGGACTTTTATATAGCATAGTTTTTAGGATCATTTCTTTGACACTA 425

TTTTATACAGATTTTAATGGGGACTAACATGAACTTTGGAAGTAACCCACCGCCGTCAGGTGATTGCGGCGGGAACAAACCGCCG 510

GGCAATAACCTGTTAACTAGACAGCCGTCAATCTATTCTCTAACCTTTGATGAGTTTCAAAGCACTATGGGTGGAATAGGCAAAG 595

ATTTTGGGTCAATGAACATGGATGAATTGTTGAGGAACATTTGGAGTGCTGAAGAAATTCAAACAATGGCTTCTTCCGGTGGTGT 680

CCTAGAGGGAAATGGAGGGTTACAAAGGCAAGGCTCTTTGACTCTGCCAAGAACACTTAGCCAGAAAACAGTTGATGAAGTCTGG 765

AAAGATATTTCAAAGGAGTATTCATTGGGGAAAGACGGTATTGGAGGTGGAGGAGGCACTAATAATATGCCACAAAGGCAGCAAA 850

CTTTAGGAGAGATGACTTTAGAGGAGTTTTTGGTGAGGGCTGGTGTGGTAAGAGAGGATACCCAATTGGCTGGGAAGGTTAATAA 935

CGGGGGGTTCTTTGGTGGGAATAATACTGGTTTTGAAATTGGGTTTCAACAAGGTGGTAAAGGTCCAAATTTGATGGGAACTAGG 1020

ATTCCTGATGGTGGTAACCAAATTGGTATTCAGGCTTCCAATTTGCATCCTAACGTTAATGGAGTTAGATCAAACCAGCACCAGT 1105

TGGCTCAGCAGCACCAACACCAACAACCAATCTTTCCTAAGCAAACAGGGGTGGGGTATGGAGCTCAGATACCTTTACAAAGTGG 1190

CGGTCAGTTGGGGAGTCCTGGAATTCGGAGTGGAATGCATGGGATTGGGGATCAGGGAATAAGTAATGGTCTGATTCAGGCAGGT 1275

GCACTGCAAGGTGGAGGCATGGGGATGGTTGGTTTAGGAACCGGATCACCTGCTAACCAGGTTTCGTCAGATGGGATTGGGAAGA 1360

GCAGTGGAGATACTTCATCAGTTTCCCCAGTTCCTTATGTGTTTAATGGAAGCATGAGGGGTAGGAAATACAGTGCGGTGGAAAA 1445

GGTTGCTGAGAGGAGGCAAAGGAGAATGATAAAGAACAGAGAATCAGCTGCAAGATCACGAGCTCGCAAGCAGGCTTATACAATG 1530

GAATTGGAAGCAGAAGTTGCAAAGCTAAAAGAGGAGAATCAAGAATTGCGGAAGAAACATGCAGAAATCATGGAAATGCAGAAAA 1615

ATCAGGTCATTGAGATGGTGGATATGCAACAAGGAGCTAAGAAGCGATGCCTACGAAGAACCCAGACCGGTCCTTGGTGAAAGTA 1700

AATCATGTTTACGAAGAAGCCTGTGATTCGTGGTTAAAACTTAAAATTATGCGTGGCAGGTGGTTGTACATATTTAAGTGGCAAG 1785

GATGGTTCCATCTTTAGGTTTGGAGTGATGAATTAGTGCTGTAGCCTGTAGTGTAGAATAAGGATTTTCCAATATTTGCAATAAT 1870

TTTTTTCCTGCTCCCCCAAGGATGATGGTTCATTTAGTTAACCACATCTGTAAATTTCCGAATATCCCCAACATCAATTGGAACT 1955

AATTTGAGTTTATTTCATGAAAAAGCATGGCCCATTTAGTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 2027

<210>2

<211>417

<212>PRT

<213>Gossypium hirsutum Y18R

<400>2

Met Gly Thr Asn Met Asn Phe Gly Ser Asn Pro Pro Pro Ser Gly Asp Cys Gly Gly Asn lys Pro 22

Pro Gly Asn Asn Leu Leu Thr Arg Gln Pro Ser Ile Tyr Ser Leu Thr Phe Asp Glu Phe Gln Ser 44

Thr Met Gly Gly Ile Gly Lys Asp Phe Gly Ser Met Asn Met Asp Glu Leu Leu Arg Asn Ile Trp 66

Ser Ala Glu Glu Ile Gln Thr Met Ala Ser Ser Gly Gly Val Leu Glu Gly Asn Gly Gly Leu Gln 88

Arg Gln Gly Ser Leu Thr Leu Pro Arg Thr Leu Ser Gln Lys Thr Val Asp Glu Val Trp Lys Asp 110

Ile Ser Lys Glu Tyr Ser Leu Gly Lys Asp Gly Ile Gly Gly Gly Gly Gly Thr Asn Asn Met Pro 132

Gln Arg Gln Gln Thr Leu Gly Glu Met Thr Leu Glu Glu Phe Leu Val Arg Ala Gly Val Val Arg 154

Glu Asp Thr Gln Leu Ala Gly Lys Val Asn Asn Gly Gly Phe Phe Gly Gly Asn Asn Thr Gly Phe 176

Glu Ile Gly Phe Gln Gln Gly Gly Lys Gly Pro Asn Leu Met Gly Thr Arg Ile Pro Asp Gly Gly 198

Asn Gln Ile Gly Ile Gln Ala Ser Asn Leu His Pro Asn Val Asn Gly Val Arg Ser Asn Gln His 220

Gln Leu Ala Gln Gln His Gln His Gln Gln Pro Ile Phe Pro Lys Gln Thr Gly Val Gly Tyr Gly 242

Ala Gln Ile Pro Leu Gln Ser Gly Gly Gln Leu Gly Ser Pro Gly Ile Arg Ser Gly Met His Gly 264

Ile Gly Asp Gln Gly Ile Ser Asn Gly Leu Ile Gln Ala Gly Ala Leu Gln Gly Gly Gly Met Gly 286

Met Val Gly Leu Gly Thr Gly Ser Pro Ala Asn Gln Val Ser Ser Asp Gly Ile Gly Lys Ser Ser 308

Gly Asp Thr Ser Ser Val Ser Pro Val Pro Tyr Val Phe Asn Gly Ser Met Arg Gly Arg Lys Tyr 330

Ser Ala Val Glu Lys Val Ala Glu Arg Arg Gln Arg Arg Met Ile Lys Asn Arg Glu Ser Ala Ala 352

Arg Ser Arg Ala Arg Lys Gln Ala Tyr Thr Met Glu Leu Glu Ala Glu Val Ala Lys Leu Lys Glu 374

Glu Asn Gln Glu Leu Arg Lys Lys His Ala Glu Ile Met Glu Met Gln Lys Asn Gln Val Ile Glu 396

Met Val Asp Met Gln Gln Gly Ala Lys Lys Arg Cys Leu Arg Arg Thr Gln Thr Gly Pro Trp 417

<210>3

<211>2177

<212>DNA

<213>Gossypium hirsutum Y18R

<400>3

ATGGGGACTAACATGAACTTTGGAAGTAACCCACCGCCGTCAGGTGATTGCGGCGGGAACAAACCGCCGGGCAATAACCTGTTAA 85

CTAGACAGCCGTCAATCTATTCTCTAACCTTTGATGAGTTTCAAAGCACTATGGGTGGAATAGGCAAAGATTTTGGGTCAATGAA 170

CATGGATGAATTGTTGAGGAACATTTGGAGTGCTGAAGAAATTCAAACAATGGCTTCTTCCGGTGGTGTCCTAGAGGGAAATGGA 255

GGGTTACAAAGGCAAGGCTCTTTGACTCTGCCAAGAACACTTAGCCAGAAAACAGTTGATGAAGTCTGGAAAGATATTTCAAAGG 340

AGTATTCATTGGGGAAAGACGGTATTGGAGGTGGAGGAGGCACTAATAATATGCCACAAAGGCAGCAAACTTTAGGAGAGATGAC 425

TTTAGAGGAGTTTTTGGTGAGGGCTGGTGTGGTAAGAGAGGATACCCAATTGGCTGGGAAGGTTAATAACGGGGGGTTCTTTGGT 510

GGGAATAATACTGGTTTTGAAATTGGGTTTCAACAAGGTGGTAAAGGTCCAAATTTGATGGGAACTAGGATTCCTGATGGTGGTA 595

ACCAAATTGGTATTCAGGCTTCCAATTTGCATCCTAACGTTAATGGAGTTAGATCAAACCAGCACCAGTTGGCTCAGCAGCACCA 680

ACACCAACAACCAATCTTTCCTAAGCAAACAGGGGTGGGGTATGGAGCTCAGATACCTTTACAAAGTGGCGGTCAGTTGGGGAGT 765

CCTGGAATTCGGAGTGGAATGCATGGGATTGGGGATCAGGGAATAAGTAATGGTCTGATTCAGGCAGGTGCACTGCAAGGTGGAG 850

GCATGGGGATGGTTGGTTTAGGAACCGGATCACCTGCTAACCAGGTTTCGTCAGATGGGATTGGGAAGAGCAGTGGAGATACTTC 935

ATCAGTTTCCCCAGTTCCTTATGTGTTTAATGGAAGCATGAGGGGTAGGAAATACAGTGCGGTGGAAAAGGTTGCTGAGAGGAGG 1020

CAAAGGAGAATGATAAAGAACAGAGAATCAGCTGCAAGATCACGAGCTCGCAAGCAGGTGAATCTATTCTCTAGGTTACTCAAAT 1105

TCGTTTAGCTACTCAATGAACTTCTGGTTGAAATTTTATGCTTTTCCAGGCCATTAAAACGGAACCCACATTAAATTTCCAAAGT 1190

TGAAAAATCAGAATTACCATCCCACCCAGTTTCCTCTTGGTGGGGGGCTTAATGAGTTTTATCAGTCATTGACCTTCTGCTCTTA 1275

TTATCTGTGACTTGGGGCGTACCGAAACTTCATGGAATATTTATGCTGCAGTATGGTAATTTTGATAGCCATCTGACATGACATG 1360

GGAAGTTTTACTAGCTAAATAAAGCATTTGTTTAACAACATGGGAAGTTTGAAGCTGTGCATCGTATACAAACCTAAAAAAACCC 1445

TTTAATTGAGGTTGACATTCTTGACCTTATTGATTTTGGTATAACTATTATCAAGCAAAACGGTTGTATCCCATGGGGGCTTTTA 1530

ACCAGGGAAATTACTGTTGTCTCAAACATGCATTGAAGTTTCTTAATTTTTTTTTTTTTTGGGGGGGGGGGGGGGGGGGGGCTTC 1615

CTAGGCTTATACAATGGAATTGGAAGCAGAAGTTGCAAAGCTAAAAGAGGAGAATCAAGAATTGCGGAAGAAACATGTATTTTAC 1700

TTTTCTTTCTTTCAATGCACCCCTTATCATTTGCACACAATAGTACTTAATTCATTCTCTTATGGTCTGTACTTTGGTTGTTTGC 1785

AGGCAGAAATCATGGAAATGCAGAAAAATCAGGTGAGGAAGTTGTTTTTGGCACTTCTGTTTTTATTTATATACTAACAACTTAA 1870

GTTATACCCTCTTCAAACACCAGGAAGCATCGTTATTTGCATAATCTTCCATTATGTACCCTATTGCACTACTTGGTTTACTTTG 1955

GAAAGCATTTTTAATTGCCCAATCCACGTGAGCGGTAAACTTTTCTTTTGCCGTATTAAATATTATGCCCGCCCTCACTAGAAAA 2040

TGTGCTTTGAACTTGGCAACCTTTGAATGTTCAATATTCTAAAATCATGCTTAATCTTGCGCAAGGTCATTGAGATGGTGGATAT 2125

GCAACAAGGAGCTAAGAAGCGATGCCTACGAAGAACCCAGACCGGTCCTTGG 2177

<210>4

<211>2056

<212>DNA

<213>Gossypium hirsutum Y18R

<220>

<221>CDS

<222>(333)..(1553)

<400>4

GTTGTTTCTGCTGTGAATGTGATGGAATTTGACGAGCATTTGTTTGGCTATGTGTATGATTGTATGTTTTGTGCTAGTTGTTAGA 85

GAATCTTGAAATTTAGAGATTTGACATGAAAGTTCTAATCTTTATTGGTTCTAATGTATGATCAAGTTACACTTTGCTTTGTTTG 170

GTGATCATTGATTAGATCTCAAGTTAAAGAAAGAAAGAAAAAAAAGTTATATCAAAGGGAAAGTGAATAATGTCATGATCATCTT 255

ATCTACTTCAGTTGATTGGATTTTATAGGGGAATTTGAATTGTGTTCTTTTGTGTTTGTTGCTGTTTGAAACAAGAAATGGGGTC 340

TAATCTGAATTTCAAGAGGTTTGGTGAAGCTCCAAGTATGGAAGGGAGTGGATCAAAGGCAGTGGGCAATTTCCCATTGGCTAGG 425

CAGTCATCGATATACTCGTTGACCTTCGATGAGCTGCAGAACACATTCGGTGGACTCGGCAAGGACTTCGGATCTATGAACATGG 510

ATGAACTCTTGAGGAACATCTCAACTGCTGAAGAGACTCATGGTTTGATGACAGCATCAGTTCCTGGAGGCGAAGGTGTTTCTGG 595

TGGTAATTTGCAGAGGCAAGGTTCATTGACATTGCCAAGGACTCTGAGTCAGAAAACGGTTGAGGAAGTATGGAAAGACTTGTTT 680

AAGGAAAATGATGGTGCTAAAAATGTAAGTAATGGTGGTGGTGGTGGTGGAGCTAATTTGCCACAGAGGCAACAGACATTGAGAG 765

AGATGACTTTGGAGGAGTTCCTGGGGAGGGCAGGTGTTGTGAGGGAAGATATGCAACCGGTTGGTGTGCCGAATAACAATGGATT 850

TTTTGATAACAACTCTGGTTTAGCCCTTCAGTTTCAACAGAGAAATGGAAACAATGGTTTTTTGAGTAACAATAACTCAGTTCTT 935

AATCAGCCTCCAATCTTACCATTAGACGTGAGTGGAGCCAAATCATCACACTCACAGCAGCAACAACAGCCACTCTTTCCCAAGC 1020

AACAAACGGTTGCATTTGCTCCATCTATGCACTTGATAAACACTACACATTTTCCTAGTCCTGGAGCTCAGGGGTCGGTAGTTGA 1105

AACCAGTGACCTTTCAATGAATACTAATCTAGTTCAGTCTAGTGGCCTGCAGAGTGGTGGGATGGGAATAGTTGGTTTACCATCT 1190

CCTGCAAGCCATATATCTCCAGATGTGATTTCAAAGAATAGTGTAGATACAACTTCTTTATCACCGGTTCCTTATTTGCTTGGCC 1275

GGGGAAGAAAACGCAGTGCAGCATTGGAGAAAGTAGTTGAGAGAAGGCAAAGGAGAATGATTAAGAACAGAGAGTCAGCTGCAAG 1360

ATCACGAGCTCGCAAGCAGGCTTATACATTGGAACTTGAAGCTGAAGTTGCAAAACTTAAAGAAATGAATGAAGAATTGCTGAGG 1445

AAACAGGAAGAAGTGATGGAAATGCAGAAAAATCAGATGCTAGAAACACTTAATCCAGCATGGGGAGGTAAAAGACAATGCTTAA 1530

GAAGAACACTGACAGGCCCTTGGTAGAGTTTGAGGAACTGCTGATTGTAAAAAATGTTGCTTTGATACACAATCTTGGTCGGTGC 1615

TACAGTTTAATGATTGGGATCTGCATCCATTATCTATATCTTGATACTGCTTAAGGCTAGATAGCTGTGAATATAGAAGCAAAGG 1700

GTTTTGAGTTGTAAGTTATGAACTTGACAGTATTAGTTTGTAGTTTTGATTTTGTTATTGGCTTGATCTCTGTTTGCCTATCATG 1785

ATCTAACCTTGACAACAAGGGCGGGCTTGTAAGATAACTAGATGGATGCTCCAATATGGTCTTTATGCTCTGCCTACAGACTTGG 1870

TCAGTTTCATCCTGATTTGACTTGTAAATTTAAGACATATCTGTTGTAAGAAATAAGCTATCGGTTTTTGATTCAAAAAAAAAAA 1955

AAAAAAA 1963

<210>5

<211>407

<212>PRT

<213>Gossypium hirsutum Y18R

<400>5

Met Gly Ser Asn Leu Asn Phe Lys Arg Phe Gly Glu Ala Pro Ser Met Glu Gly Ser Gly Ser Lys 22

Ala Val Gly Asn Phe Pro Leu Ala Arg Gln Ser Ser Ile Tyr Ser Leu Thr Phe Asp Glu Leu Gln 44

Asn Thr Phe Gly Gly Leu Gly Lys Asp Phe Gly Ser Met Asn Met Asp Glu Leu Leu Arg Asn Ile 66

Ser Thr Ala Glu Glu Thr His Gly Leu Met Thr Ala Ser Val Pro Gly Gly Glu Gly Val Ser Gly 88

Gly Asn Leu Gln Arg Gln Gly Ser Leu Thr Leu Pro Arg Thr Leu Ser Gln Lys Thr Val Glu Glu 110

Val Trp Lys Asp Leu Phe Lys Glu Asn Asp Gly Ala Lys Asn Val Ser Asn Gly Gly Gly Gly Gly 132

Gly Ala Asn Leu Pro Gln Arg Gln Gln Thr Leu Arg Glu Met Thr Leu Glu Glu Phe Leu Gly Arg 154

Ala Gly Val Val Arg Glu Asp Met Gln Pro Val Gly Val Pro Asn Asn Asn Gly Phe Phe Asp Asn 176

Asn Ser Gly Leu Ala Leu Gln Phe Gln Gln Arg Asn Gly Asn Asn Gly Phe Leu Ser Asn Asn Asn 198

Ser Val Leu Asn Gln Pro Pro Ile Leu Pro Leu Asp Val Ser Gly Ala Lys Ser Ser His Ser Gln 220

Gln Gln Gln Gln Pro Leu Phe Pro Lys Gln Gln Thr Val Ala Phe Ala Pro Ser Met His Leu Ile 242

Asn Thr Thr His Phe Pro Ser Pro Gly Ala Gln Gly Ser Val Val Glu Thr Ser Asp Leu Ser Met 264

Asn Thr Asn Leu Val Gln Ser Ser Gly Leu Gln Ser Gly Gly Met Gly Ile Val Gly Leu Pro Ser 286

Pro Ala Ser His Ile Ser Pro Asp Val Ile Ser Lys Asn Ser Val Asp Thr Thr Ser Leu Ser Pro 308

Val Pro Tyr Leu Leu Gly Arg Gly Arg Lys Arg Ser Ala Ala Leu Glu Lys Val Val Glu Arg Arg 330

Gln Arg Arg Met Ile Lys Asn Arg Glu Ser Ala Ala Arg Ser Arg Ala Arg Lys Gln Ala Tyr Thr 352

Leu Glu Leu Glu Ala Glu Val Ala Lys Leu Lys Glu Met Asn Glu Glu Leu Leu Arg Lys Gln Glu 374

Glu Val Met Glu Met Gln Lys Asn Gln Met Leu Glu Thr Leu Asn Pro Ala Trp Gly Gly Lys Arg 396

Gln Cys Leu Arg Arg Thr Leu Thr Gly Pro Trp 407

<210>6

<211>1773

<212>DNA

<213>Gossypium hirsutum Y18R

<400>6

ATGGGGTCTAATCTGAATTTCAAGAGGTTTGGTGAAGCTCCAAGTATGGAAGGGAGTGGATCAAAGGCAGTGGGCAATTTCCCAT 85

TGGCTAGGCAGTCATCGATATACTCGTTGACCTTCGATGAGCTGCAGAACACATTCGGTGGACTCGGCAAGGACTTCGGATCTAT 170

GAACATGGATGAACTCTTGAGGAACATCTCAACTGCTGAAGAGACTCATGGTTTGATGACAGCATCAGTTCCTGGAGGCGAAGGT 255

GTTTCTGGTGGTAATTTGCAGAGGCAAGGTTCATTGACATTGCCAAGGACTCTGAGTCAGAAAACGGTTGAGGAAGTATGGAAAG 340

ACTTGTTTAAGGAAAATGATGGTGCTAAAAATGTAAGTAATGGTGGTGGTGGTGGTGGAGCTAATTTGCCACAGAGGCAACAGAC 425

ATTGAGAGAGATGACTTTGGAGGAGTTCCTGGGGAGGGCAGGTGTTGTGAGGGAAGATATGCAACCGGTTGGTGTGCCGAATAAC 510

AATGGATTTTTTGATAACAACTCTGGTTTAGCCCTTCAGTTTCAACAGAGAAATGGAAACAATGGTTTTTTGAGTAACAATAACT 595

CAGTTCTTAATCAGCCTCCAATCTTACCATTAGACGTGAGTGGAGCCAAATCATCACACTCACAGCAGCAACAACAGCCACTCTT 680

TCCCAAGCAACAAACGGTTGCATTTGCTCCATCTATGCACTTGATAAACACTACACATTTTCCTAGTCCTGGAGCTCGGGGGTCG 765

GTAGTTGAAACCAGTGACCTTTCAATGAATACTAATCTAGTTCAGTCTAGTGGCCTGCAGAGTGGTGGGATGGGAATAGTTGGTT 850

TACCATCTCCTGCAAGCCATATATCTCCAGATGTGATTTCAAAGAATAGTGTAGATACAACTTTATCACCGGTTCCTTATTTGCT 935

TGGCCGGGGAAGAAAACGCAGTGCAGCATTGGAGAAAGTAGTTGAGAGAAGGCAAAGGAGAATGATTAAGAACAGAGAGTCAGCT 1020

GCAAGATCACGAGCTCGCAAGCAGGTGAGTTGAGTGCCACTACACTGTTGTTTCCTTTCCTTCTGAGTTACCAGATCCTATTTCA 1105

TTTCTCCTATAAAATCTTGCTCAGTTGTGTGGATTACTTGTTTAAAAAGTTCATTAGTTTTGAATTTTGTTGCTTTCTACAAACA 1190

TGCCACTCATAATGTTTAGGACAATGCCCCTACATTGCTTGTGTGAAAGTAATGGTTTGGTATGGTTTATAGTTGGCTCAAATCC 1275

AATGCCAAACAAGATTGTATTAATTGCTCTGAGTTCTGGCAGGCTTATACATTGGAACTTGAAGCTGAAGTTGCAAAACTTAAAG 1360

AAATGAATGAAGAATTGCTGAGGAAACAGGTAAGTACTTATCACCTGGACTTCGATTCATCGTATTCTATTGAAACGTTGGTGGA 1445

TCGGCCCTTTTCTTGACAGTGCTTTGGTTGGTGCAGGAAGAAGTGATGGAAATGCAGAAAAATCAGGTAATGCAACATTCCTGCT 1530

GCTTCCATCTTGCATTTATGTTATAGCATTCATATGCAGCAAACTAGCTTTTAGATGGGTTCCAAAGCCTTAAGAACATGGCAAA 1615

CCATGATATATAACATGTTTTGTTCCATGCACATTTATTGAAATGCTTCCTGTTTTTGGTCTGCTCTCTGCTGCCTTTTCAAACA 1700

GATGCTAGAAACACTTAATCCAGCATGGGGAGGTAAAAGACAATGCTTAAGAAGAACACTGACAGGCCCTTGG 1773

<210>7

<211>1927

<212>DNA

<213>Gossypium hirsutum Y18R

<220>

<221>CDS

<222>(384)..(1649)

<400>7

AGTGAGAAGAACAAAAGAGGCAGCAGCAGCAGCACACGCGTTGAGCTAAGAGCAGTAAAGCAAAGCAGAGCAAGCCACATGGCCG 85

AAACGTGTCGATCAAAGCAACGCCGATACCATTTTGGAACTGGGGAGACAGCACAATTACCCAATTTTTATAAAGTTTTCCCTTC 170

CTTTGGGTCGATTCCAATTGACTTTTTTGGACCCCACTGCGCCGGGAAACTTTAAACTCACTTTTACACCCTCTACGTGTTTGTT 255

TATATTCCCCACTGCCTAACCATTTTCTTCAACCACAATAACCCTCTTATTTTTTCATCTTTTTTCAGCTCCAATCATATCTCTT 340

TTCCTTTGTTGAAACCAGTTTAAAGCAAGATTTCATCGCAAAAATGGTGGTTGAGAACTCTGAGGTTGGCGAGGTCGAGTCCACA 425

TTGAAGGAGGTGGACCAGCAGCTAAAGAATCATCCATTATCGGCCCTTGGAAGACAATCCTCAATTTATTCCCTCACTCTCGATG 510

AGTTCCAGCACACGTTATGTGGGAAGAACTTTGGGTCGATGAACATGGACGAGTTCCTTACCAGCATTTGGAATGCTGAAGAAAA 595

CCAAGCTACAAACTCCATTAATAACAATCATCACAACAATGGTGTAAATGAACAGGTTAGCAATCATGTTAACTTATCTTTGAAT 680

GAAACAGCAAGCAGTAAAGGTGTAGCTAGGCAGTCTAGCTTGCCTCGACAAGGCTCGCTTTCACTTCCGGCACCATTGTGTCGGA 765

AAACTGTCGATGAAGTTTGGTCTGAGATACATAAAGTGCAGCAAGGACAAGGACAGAGTAACAAGAGCAATGTGCAGAATGCTGA 850

GAATACTAGTACTCGACAACCGACTTTTGGGGAAATGACCTTGGAAGATTTCTTGGTTAAGGCAGGTGTAGTTCGGGAACCATGC 935

GTACCACCGGCAGTACCGCCGCATTCACAACATCAACAACAGTTCGGGTTGCATCAGGCTAGCAATAACCCTGCAGTGGGTCCGA 1020

GTTTTGTTCCCAGGCCAATTATGGGAATGGGTGGCAGTGGTGGCTTTGGTGGGAGTACATATCAAACAATGCCTCCAAGTGGAGT 1105

CCTTGGTGATTCATCACGCTATCTTAATGATGGTAAGGGGGGTGGCGGCTACCAGCCAGCTGCTGCCCCACCGTCAACAGCCATC 1190

TGTTATAACGGAAAAGTAGGTGCAGCTGGTGGCTTTGGACCGGGACAGGCAATGGGAGTGGTATCACCAATAAGTCCGGTATCAC 1275

CAGATGGGATATGTACCAATCAAGTTGATAATGCAGCCACTCAATTCGGGATAGAGATAGGTGGACTACGAGGAAGGAAAAGAAT 1360

CATTGATGGTCCAATAGAAAAAGTAGTCGAGAGAAGGCAACGCAGGATGATTAAGAATAGGGAATCAGCTGCAAGGTCTAGAGCT 1445

AGAAAACAGGCATATACAGTTGAGCTAGAAGCAGAATTGAACCAATTGAAACAAGAGAATACTCACCTCAAGCAGGCTTTGGTGG 1530

AACTCGAGAGGAAAAGAAAGCAACAGTACTTTGAGGAATGGTCAATGAAAACACAAACCAAAGCCCAGAAAGCTAAAGAGAAACT 1615

AAGGGTAATGAGAAGAAATCTAAGTGGTCCATTATGAGCCAGAAAAGAAGATGCATGCTCAAGGCATTGACGGTGGAAGCTCTCA 1700

AAACCAATGACATAACCTCAAGCTGAAGAACATCATCAAAACACGTTTAGACACATGATAGTTGGTGGATATTTATAAAGAAGGC 1785

TTCTGGGAATATGATTTTTATGAATAATCAAATTTCTGAAGTCTAAACTAATGATATATATATGTATGTATGTATACGGTTTTAA 1870

TGTTCACACAAGTCATGGAAAGGTATGTCGTTGCTTGTAAAAAAAAAAAAAAAAAAA 1927

<210>8

<211>422

<212>PRT

<213>Gossypium hirsutum Y18R

<400>8

Met Val Val Glu Asn Ser Glu Val Gly Glu Val Glu Ser Thr Leu Lys Glu Val Asp Gln Gln Leu 22

Lys Asn His Pro Leu Ser Ala Leu Gly Arg Gln Ser Ser Ile Tyr Ser Leu Thr Leu Asp Glu Phe 44

Gln His Thr Leu Cys Gly Lys Asn Phe Gly Ser Met Asn Met Asp Glu Phe Leu Thr Ser Ile Trp 66

Asn Ala Glu Glu Asn Gln Ala Thr Asn Ser Ile Asn Asn Asn His His Asn Asn Gly Val Asn Glu 88

Gln Val Ser Asn His Val Asn Leu Ser Leu Asn Glu Thr Ala Ser Ser Lys Gly Val Val Arg Gln 110

Ser Ser Leu Pro Arg Gln Gly Ser Leu Ser Leu Pro Ala Pro Leu Cys Arg Lys Thr Val Asp Glu 132

Val Trp Ser Glu Ile His Lys Val Gln Gln Gly Gln Gly Gln Ser Asn Lys Ser Asn Val Gln Asn 154

Ala Glu Asn Thr Ser Thr Arg Gln Pro Thr Phe Gly Glu Met Thr Leu Glu Asp Phe Leu Val Lys 176

Ala Gly Val Val Arg Glu Pro Cys Val Pro Pro Ala Val Pro Pro His Ser Gln His Gln Gln Gln 198

Phe Gly Leu His Gln Ala Ser Asn Asn Pro Ala Val Gly Pro Ser Phe Val Pro Arg Pro Ile Met 220

Gly Met Gly Gly Ser Gly Gly Phe Gly Gly Ser Thr Tyr Gln Thr Met Pro Pro Ser Gly Val Leu 242

Gly Asp Ser Ser Arg Tyr Leu Asn Asp Gly Lys Gly Gly Gly Gly Tyr Gln Pro Ala Ala Ala Pro 264

Pro Ser Thr Ala Ile Cys Tyr Asn Gly Lys Val Gly Ala Ala Gly Gly Phe Gly Pro Gly Gln Ala 286

Met Gly Val Val Ser Pro Ile Ser Pro Val Ser Pro Asp Gly Ile Cys Thr Asn Gln Val Asp Asn 308

Ala Ala Thr Gln Phe Gly Ile Glu Ile Gly Gly Leu Arg Gly Arg Lys Arg Ile Ile Asp Gly Pro 330

Ile Glu Lys Val Val Glu Arg Arg Gln Arg Arg Met Ile Lys Asn Arg Glu Ser Ala Ala Arg Ser 352

Arg Ala Arg Lys Gln Ala Tyr Thr Val Glu Leu Glu Ala Glu Leu Asn Gln Leu Lys Gln Glu Asn 374

Thr His Leu Lys Gln Ala Leu Val Glu Leu Glu Arg Lys Arg Lys Gln Gln Tyr Phe Glu Glu Trp 396

Ser Met Lys Thr Gln Thr Lys Ala Gln Lys Ala Lys Glu Lys Leu Arg Val Met Arg Arg Asn Leu 418

Ser Gly Pro Leu 422

<210>9

<211>1767

<212>DNA

<213>Gossypium hirsutum Y18R

<400>9

ATGGTGGTTGAGAACTCTGAGGTTGGCGAGGTCGAGTCCACATTGAAGGAGGTGGACCAGCAGCTAAAGAATCATCCATTATCGG 85

CCCTTGGAAGACAATCCTCAATTTATTCCCTCACTCTCGATGAGTTCCAGCACACGTTATGTGGGAAGAACTTTGGGTCGATGAA 170

CATGGACGAGTTCCTTACCAGCATTTGGAATGCTGAAGAAAACCAAGCTACAAACTCCATTAATAACAATCATCACAACAATGGT 255

GTAAATGAACAGGTTAGCAATCATGTTAACTTATCTTTGAATGAAACAGCAAGCAGTAAAGGTGTAGCTAGGCAGTCTAGCTTGC 340

CTCGACAAGGCTCGCTTTCACTTCCGGCACCATTGTGTCGGAAAACTGTCGATGAAGTTTGGTCTGAGATACATAAAGTGCAGCA 425

AGGACAAGGACAGAGTAACAAGAGCAATGTGCAGAATGCTGAGAATACTAGTACTCGACAACCGACTTTTGGGGAAATGACCTTG 510

GAAGATTTCTTGGTTAAGGCAGGTGTAGTTCGGGAACCATGCGTACCACCGGCAGTACCGCCGCATTCACAACATCAACAACAGT 595

TCGGGTTGCATCAGGCTAGCAATAACCCTGCAGTGGGTCCGAGTTTTGTTCCCAGGCCAATTATGGGAATGGGTGGCAGTGGTGG 680

CTTTGGTGGGAGTACATATCAAACAATGCCTCCAAGTGGAGTCCTTGGTGATTCATCACGCTATCTTAATGATGGTAAGGGGGGT 765

GGCGGCTACCAGCCAGCTGCTGCCCCACCGTCAACAGCCATCTGTTATAACGGAAAAGTAGGTGCAGCTGGTGGCTTTGGACCGG 850

GACAGGCAATGGGAGTGGTATCACCAATAAGTCCGGTATCACCAGATGGGATATGTACCAATCAAGTTGATAATGCAGCCACTCA 935

ATTCGGGATAGAGATAGGTGGACTACGAGGAAGGAAAAGAATCATTGATGGTCCAATAGAAAAAGTAGTCGAGAGAAGGCAACGC 1020

AGGATGATTAAGAATAGGGAATCAGCTGCAAGGTCTAGAGCTAGAAAACAGGTACTACCTACAACTACTGTTTGCTGGAAGTGAA 1105

TGCATATATATGCATGTGTATACAATTATACATATATATTTTCTGGTAGAGAAATAATCTTCTGTTGTGTAGGTCATTAGTATAT 1190

AGGCATAATACTTCAATTCTTACTAGGTTGGTTATATTTAACAGGCATATACAGTTGAGCTAGAAGCAGAATTGAACCAATTGAA 1275

ACAAGAGAATACTCACCTCAAGCAGGCTTTGGTGACATTCGTATTACTTTTCGCATTTATCAAAATTTCAGATCTTGATGTAGCT 1360

TTTATCATATATAATATGGCAGCTTAATCTCATTTTACTTACATTTCAACAGGTGGAACTCGAGAGGAAAAGAAAGCAACAGGTA 1445

AATCATTCAACTGATTACATGTGCTTTACAATGCACCATATGAAGTAGAGAATACAAAAAGATGATCAGAAGGGCAAAGTTATAG 1530

AACTTTATGATGGGGGTAACATGCATAAGAGTAGGAAATATAAGCATTGATGGCTACAAGCATCATACTAGATTCATTTTTTTGC 1615

TTTGTTCATCCTATTTTTGGTGTTTTTTTTCTTTTTCTTTTTCTTTTTTGTCCTTGCAGTACTTTGAGGAATGGTCAATGAAAAC 1700

ACAAACCAAAGCCCAGAAAGCTAAAGAGAAACTAAGGGTAATGAGAAGAAATCTAAGTGGTCCATTA 1767

Claims (2)

1. cotton bZIP class transcription factor, its aminoacid sequence is shown in SEQ ID NO:2.

2. the encoding gene of the described cotton bZIP of claim 1 class transcription factor, its base sequence is shown in SEQ ID NO:3.

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