CN105073772A

CN105073772A - Myb transcription factor myb1-1 of cotton and coding gene, and use thereof

Info

Publication number: CN105073772A
Application number: CN201380074522.XA
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: 2013-04-24
Filing date: 2013-04-24
Publication date: 2015-11-18
Also published as: WO2014172852A1

Abstract

Disclosed are an MYB transcription factor MYB1-1 derived from cotton and a coding gene thereof, and the use thereof in the cultivation of transgenic plants having improved drought tolerance.

Description

Myb transcription factor myb1-1 of cotton and coding gene, and use thereof

One cotton MYB class transcription factor MYB1-1 and its encoding gene and application

The present invention relates to MYB classes transcription factor and its encoding gene and application, the more particularly to one MYB class transcription factor MYB1-1 and its encoding gene from cotton, and its application in the genetically modified plants that drought tolerance is improved are cultivated for technical field.Technical background abiotic stress, such as arid, salt marsh, extreme temperature, chemical contamination and oxygen injury can cause serious harm to growing for plant, extreme loss is caused to crop yield, wherein influence of the arid to crop yield, first place is accounted in many natural adverse circumstances, it is endangered equivalent to other disaster sums, be many areas be agricultural development bottleneck.According to statistics, the dry early, semiarid zone in the world accounts for the 34% of land area;China is dry early, semiarid zone accounts 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 300 receives 350 400 hundred million kilograms of grain because of water shortage less；Particularly China relationship and primary grain producing such as North China, northeast and northwest, is the area of China's water shortage most serious, and spring drought frequently reaches 10 years nine chances.

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

It is the key link of plant Stress response reaction by the expression of transcription factor adjusting function gene in plant stress-resistance reaction system.As one of transcription factor family maximum in plant, MYB classes transcription factor plays an important role in plant stress-resistance stress procedure.But for current research situation, because its mechanism is sufficiently complex, many plants still need to be studied to the biochemistry under adverse circumstance and physiological response mechanism.In the research in terms of the function and expression regulation of degeneration-resistant response gene important basis is provided by the research that network system is transmitted in the contact between the signaling pathways related to plant stress-resistance and whole signal.Content of the invention the present inventor utilizes SSH (Subtractive hybridizations）With RACE (cDNA ends rapid amplifyings）The method being combined has cloned a MYB class transcription factor for cotton（Be named as MYB1-1 herein) encoding gene.And find to be conducted into after recipient plant, the drought tolerance of transfer-gen plant is can obviously improve, and these characters can stablize heredity.

First aspect present invention provides a MYB class transcription factors MYB1-1 for cotton encoding gene（G/z B-are 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 inserted into a kind of expression vector by the gene and obtained, 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 rd29A- GhMYB 1-1-2300 carriers shown in accompanying drawing 2.

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

Fourth aspect present invention provides a kind of method of improvement drought resistance in plants, including：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 tobacco.

Fifth aspect present invention provides a kind of method of prepare transgenosis plant, including：Cultivated under conditions of plant is effectively produced containing the gene described in first aspect present invention, the load of the recombination expression described in second aspect of the present invention The plant of body or plant tissue；Preferably, the plant is tobacco.

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

Seventh aspect present invention provides the amino acid sequence of the gene code as described in first aspect present invention, such as SEQ ID NO:Shown in 1.

Brief description of the drawings Fig. 1 is G/z ZBl-1 plant expression vector Crd29A- GhMYBl-l-2300;>Structure flow（Scheme la-lb).

Fig. 2 is the plasmid figure of GhMYBW plant expression vector (rd29A- GhMYBl-1-2300).Fig. 3 is GhMYBl-l transgene tobaccos T_QFor plant（In figure, T_QC_{2 ;}The right side, T_QC₇) and be used as control non-transgenic tobacco plants（Figure is left, CK) drought-enduring simulated experiment result.

Fig. 4 is using reverse transcription PCR to T_QThe result of molecular level detection is carried out for the transcriptional level of GhMYBl-l genes in transgenic tobacco plant and non-transgenic reference plant.M is Marker, and 1-5 is non-transgenic reference tobacco plant, and 6-18 is the notable transgene tobacco T of drought-enduring effect_QFor plant, 19-24 is the inapparent transgene tobacco T of drought-enduring effect_QFor plant.

The present invention is further described with reference to non-limiting example for embodiment.

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

Specific method is：

According to the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kit specifications builds subtracted library by Subtractive hybridization method.The mRNA of the cotton seedling leaf of Osmotic treatment is used as sample using in growth course in experimentation（), tester the mRNA using untreated cotton seedling leaf is used as control（driver).Comprise the following steps that： (1) material to be tested：

(National Cotton mid-term storehouse obtains unit Cotton research institute, Unified number to Ji cotton 14：ZM-30270) it is seeded on the vermiculite of sterilizing, is cultivated under 25 °C, 16 hours photoperiods illumination/8 hour dark condition, 1/2MS culture mediums are poured weekly（9.39 mM KN0₃, 0.625 mM KH₂P0₄, 10.3 mM H₄N0₃, 0.75 mM MgS0₄, 1.5 mM CaCl₂, 50 μ Μ KI, Ι Ο Ο μ Μ H₃B0₃, Ι Ο Ο μ Μ MnS0₄, 30 μ Μ ZnS0₄, 1 μ Μ Ν α₂Μο0₄, O. ^M CoCl₂, ΙΟΟμΜ Na₂EDTA, 100 M FeSO₄)-secondary.It is used to test when seedling plant height reaches 25-30cm.

(2) material process：

2 groups, every group of 4 basins, per 1 plant of basin will be divided into for examination seedling.First group is control group, in 25 °C, illumination cultivation, is just commonly using 1/2MS and is pouring；Second group is Osmotic treatment group, the culture under 25 °C, 16 hours photoperiods illumination/8 hour dark condition, stops pouring, the blade of timely two groups of seedling apicals 1/3 of clip after 10 days, after liquid nitrogen quick freeze, preserved in -70 °C of refrigerators.

(3) Total RNAs extraction：

Control group and the cotton leaf 0.5g of Osmotic treatment group are taken respectively, and the total serum IgE of cotton is extracted with plant RNA extracts kit (Invitrogen).Total serum IgE is determined in 260 nm and 280 nm absorbance with the ultraviolet specrophotometer U-2001 of HITACHI companies, OD260/OD280 ratios are 1.8-2.0, show that total serum IgE purity is higher, the integrality of total serum IgE is detected with 1.0% agarose gel electrophoresis, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.Use the Oligotex mRNA purification kits of Qiagen companies（PolyA+ RNA are purified from total serum IgE) separation mRNA.

(4) Subtractive hybridization：

In order to have increased access to EST（Expressed sequence tag, EST) (unigene) validity, it is to avoid gene is without restriction enzyme site and obtained sequence in non-translational region.This laboratory divides another ij to bend I things 01igo (dT) according to the scheme described in subtractive kit by two groups of mRNA₁₈(TTTTTTTTTTTTTTTTTT) reverse transcription is carried out, and gained cDNA Rsal, Haelll are digested, two groups of suppression subtractives are done, other steps and method press the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kits carries out Subtractive hybridization, finally merges second of PCR product that two groups of positive subtractives hybridize cDNA fragments.

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

The positive subtractive of merging is hybridized to second of PCR primer of cDNA fragments（QIAquick PCR Purification Kit are purified, purchased from Qiagen) it is connected with pGEM-T Easy (being purchased from Promega kits) carrier, according to method shown in pGEM-T Easy reagent kit product specifications, comprises the following steps that：Following ingredients are sequentially added with 200 μ PCR pipes：The positive subtractive of purifying hybridizes the μ of 4 DNA ligase of 4 ligase buffer solution of second of PCR primer 3 μ 1, Τ 5 μ 1, pGEM-T Easy carriers 1 μ 1, Τ 1 of cDNA fragments, is stayed overnight in 4 °C of connections.Then 10 coupled reaction products are taken, are added in 100 competence Escherichia coli JMI09 (being purchased from TAKARA), the min of ice bath 30, the s of heat shock 60, the min of ice bath 2 separately add 250 μ L LB fluid nutrient mediums（Contain 1% tryptone（Tryptone, purchased from OXOID), the NaCl of 0.5% yeast extract (Yeast Extract, purchased from OXOID) standing grain P 1% (be purchased from traditional Chinese medicines））After be placed in 37 °C of shaking tables, with the min of 225 r/min shaken cultivations 30,200 μ bacterium solutions are then therefrom taken to plant in containing 50 g/mL ampicillins, the g/mL IPTG of 40 igl L X-gaK 24 (X-gal (the chloro- 3- indoles-β-D- galactosides of 5- bromo- 4）With IPTG (isopropyl-β-D- Thiogalactopyranosides）Purchased from TAKARA) LB (ibid）On solid culture flat board, 37 °C of 18 h of cultivation.Count diameter in culture plate>L mm clear white and blue colonies number, random 360 white colonies of picking (numbering：Gh-D001 to Gh-D360).The white colony of institute's picking is inoculated in after the LB fluid nutrient mediums containing 50 g/mL ampicillins in 96 porocyte culture plates (CORNING), 37 °C of overnight incubations glycerol adding to the (volume ratio of final glycerol concentration 20%）, saved backup in -80 °C.To the colony clone cultivated with nest-type PRC bow ^ Primer 1 and Primer 2R (the PCR-selectTM cDNA Subtraction Kit kits from Clontech companies）Bacterium solution PCR amplification checkings are carried out, 292 positive colonies is obtained, Invitrogen is being sent to all positive colonies（Shanghai）Trade Co., Ltd is sequenced.

(6) the cDNA sequencing analysis of differential cloning：

After the cDNA that DNA sequencing result is removed to carrier and indefinite sequence and redundancy, 180 ESTs are obtained（Expressed sequence tag, EST) (unigene).Find that wherein 102 unigene have homologous sequence in GenBank through BlastN（Albumen homology more than 50%）, 33 EST Unknown Functions or for assume albumen, separately there are 45 not obtain homologous matching, thus it is speculated that to be probably the shorter sequence in 3 ', 5 ' end non-translational regions.The cotton MYB class transcription factor encoding genes GhMYBl-l of embodiment 2 clone

One of effective clone that embodiment 1 is obtained Gh-D178 sequencing result removes after redundant DNA, sequence is SEQ ID N0 3, sequence analysis shows that the amino acid sequence of the coding of the sequence and known MYB classes transcription factor sequence have higher homology, and the full-length gene for herein encoding clone Gh-D178 is named MYB1-1 is named as the corresponding albumen of GhMYBW.

SEQ ID NO:3

1 GCTGCTGGCC TTCTCCGTTG TGGCAAAAGT TGCAGGCTTC GTTGGATCAA CTACTTAAGA

61 CCTGACCTCA AACGTGGCAA CTTCACTGAG GAAGAAGATG AACTCATTAT CAAGCTCCAT 121 AGCCTTCTCG GCAACAAGTG GTCTCTTATA GCCGGGAGAT TACCAGGAAG AACGGATAAT

181 GAGATAAAGA ATTACTGGAA CACACACATA AGGAGGAAGC TATTGAACAG AGGAATCGAT

241 CCAGCGACTC ACAGGCCACT GAGTGAGGCA GGTGTTCATC AGGATGTGTC ATCCACAATA

301 TCCTTCAATG GTGTTAAACA AGAAAATGAC AAGATGAATA GCCCCAATGG TTTTGTGGAG

361 ACAGATGAGA AGAAAATCCC AGCAGTTGAA GAAAGGTGTC CAGACTTGAA TTTGGACCTA 421 AGAATCAGCC CACCTCCTTA CCATCAAACC CAGCAGCACG CAGATCCATT CAAAACTGGA

481 GGGAAAACCC TCTGTTTATT TTGCAGTTTG GGAGTTAAAA ACAGCAAACA ATGCATTTGC

541 AGCATCGATA CTGCTGCCAG CAGCAGTGGC AACAACACCA ATACTGCCTA TGATTTCTTA

601 GGCTTGAAAA CTGGTTTCTT GGATTACAGA AGTTTGGAAA TGAAATGATT GTTTCAGAGG

661 GAAATGTGGA ATTTTTTTTC CTTCTTTTTT TAACCATGTA GCTTAAGCAG AAATGGAGAG 721 AATTAATTGA CCA

The clone of GhMYBl-l full-length genes

The GhMYBl-l genetic fragments obtained（SEQ ID NO:3), there is terminator codon TGA, it is only necessary to be 5 ' RACE.

According to the G Sl-1 genetic fragments obtained（SEQ ID NO:3) three specific primers, are designed, reverse transcription primer and 5'RACE 3 ' end primers are used as.

GhMYBl-l GSP1 : SEQ ID NO: 4：

GTTGTTGCCACTGCTGCTGG

GhMYBl-l GSP2: SEQ ID NO: 5：

CTGCAAAATAAACAGAGGGT

GhMYBl-l GSP3 : SEQ ID NO: 6：

TGCTGGGTTTGATGGTAAGG

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

With SEQ ID NO:5 (kit is carried with 5' universal primers AAP）, cDNA (the reverse transcription primer SEQ ID NO for the mRNA reverse transcriptions extracted with Osmotic treatment group cotton leaf:4) first round is carried out for template

PCR is expanded, and is comprised the following steps that：Ex Taq are purchased from TAKARA, 50 μ PCR reaction systems： 5 μΐ ΙΟ Εχ

Buffer, 3 μ 2.5 mM the μ Ex of dNTP, the cDNA of 2.0 μm of RNA reverse transcriptions, 1.0 Taq, 10 μ Μ primer SEQ ID NO:Each 2.0 μ 1 of 5 and AAP, and 35 μ distilled water.PCR reaction conditions：

94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 55 °C of annealing 30 s, 72 °C of extension 2min, after 33 circulations, 72 °C of 10 min of extension.The PCR primer of gained is diluted with distilled water takes 2.0 μ as template after 50 times, use

SEQ ID NO:6 carry out second with 3' ends primer AUAP takes turns PCR amplifications, comprises the following steps that：50 μ PCR reaction systems：The first round PCR product of 5 μ Ι Ο Ε χ Buffer, 3 μ 2.5 mM dNTP, 2.0 μ dilution, 1.0 μ Ex Taq, 10 μ Μ primer SEQ ID NO:Each 2.0 μ 1 of 6 and AUAP, and 35 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of 30 s of annealing,

72 °C of extension 2min, after 33 circulations, 72 °C of 10 min of extension.It is about 600bp bands that second of PCR primer, which reclaims fragment,（Gel Extraction Kit are purchased from OMEGA) it is connected to pGEM-T Easy carriers, it is transformed into JM109 (specific method is ibid), random 10 white colonies of picking are cultivated in the LB fluid nutrient mediums containing 50 g/mL ampicillins, and glycerol adding to final glycerol concentration is 20% (volume ratio after 37 °C of overnight incubations）, -80 °C save backup. SEQ ID NO:6 carry out bacterium solution PCR amplifications with 3' ends primer AUAP（Reaction system and reaction condition are ibid）, 3 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd is sequenced, and obtains the cDNA of gene 5' ends.After the 5'RACE product clonings sequencing of gained, splice with clone Gh-D178 sequencing results, obtain

SEQ ID NO: 17：

1 CACCAAACTC CACCCTTCTT CGTTTCCGTT TCCCTACTGC TGCTTCTATT TTCTAAATCC

61 CAAAATGGGA AGGTCTCCTT GCTGTGAGAA AGCTCATACA AACAAAGGTG CATGGACCAA

121 AGAAGAAGAT GATCGCCTCA TCGCTTACAT CCGAGCCCAC GGTGAAGGTT GCTGGCGTTC

181 TCTCCCTAAG GCTGCTGGCC TTCTCCGTTG TGGCAAAAGT TGCAGGCTTC GTTGGATCAA 241 CTACTTAAGA CCTGACCTCA AACGTGGCAA CTTCACTGAG GAAGAAGATG AACTCATTAT

301 CAAGCTCCAT AGCCTTCTCG GCAACAAGTG GTCTCTTATA GCCGGGAGAT TACCAGGAAG

361 AACGGATAAT GAGATAAAGA ATTACTGGAA CACACACATA AGGAGGAAGC TATTGAACAG

421 AGGAATCGAT CCAGCGACTC ACAGGCCACT GAGTGAGGCA GGTGTTCATC AGGATGTGTC

481 ATCCACAATA TCCTTCAATG GTGTTAAACA AGAAAATGAC AAGATGAATA GCCCCAATGG 541 TTTTGTGGAG ACAGATGAGA AGAAAATCCC AGCAGTTGAA GAAAGGTGTC CAGACTTGAA

601 TTTGGACCTA AGAATCAGCC CACCTCCTTA CCATCAAACC CAGCAGCACG CAGATCCATT

661 CAAAACTGGA GGGAAAACCC TCTGTTTATT TTGCAGTTTG GGAGTTAAAA ACAGCAAACA

721 ATGCATTTGC AGCATCGATA CTGCTGCCAG CAGCAGTGGC AACAACACCA ATACTGCCTA

781 TGATTTCTTA GGCTTGAAAA CTGGTTTCTT GGATTACAGA AGTTTGGAAA TGAAATGATT 841 GTTTCAGAGG GAAATGTGGA ATTTTTTTTC CTTCTTTTTT TAACCATGTA GCTTAAGCAG

901 AAATGGAGAG AATTAATTGA CCA

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

GhMYBl-rp： SEQ ID NO: 7：

ATGGGAAGGTCTCCTTGCTGTG

GhMYBl-l : SEQ ID NO: 8： TCATTTCATTTCCAAACTTCTG

Pass through SEQ ID NO:7 and SEQ ID NO:8 clone GhMYBW total lengths.

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, the cDNA for the mRNA reverse transcriptions extracted using Osmotic treatment group cotton leaf enters performing PCR reaction as template.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 2.0 μ cDNA, 1.0 μ PrimeSTAR, 10 μ Μ primer

SEQ ID NO:7 and SEQ ID NO:8 each 2.0 μ 1, and 30 μ distilled water.PCR reaction conditions：

94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, after 33 circulations,

72 °C of 10 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 are placed 10 minutes, centrifugation, supernatant is removed, is dried, is dissolved with 21 μ distilled waters, add 2.5 μ Ι Ο χ Ε χ Buffer, 0.5 μ 15 ι η Μ dATP, 2.5 μ Ι Ο χ Ε χ Taq.Reaction condition：70 °C are reacted 30 minutes.The DNA fragmentation for obtaining about 800 bp is reclaimed（Omega QIAquick Gel Extraction Kits）It is connected on pGEM T-easy carriers, JM109 (method is ibid) is converted, random 10 white colonies of picking cultivate in the LB fluid nutrient mediums containing 50 g/mL ampicillins, glycerol adding is to (the volume ratio of final glycerol concentration 20% after 37 °C of overnight incubations）, -80 °C save backup. SEQ ID NO:7 and SEQ ID NO:8 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 4 positive colonies are obtained, Invitrogen is delivered to（Shanghai）Trade Co., Ltd is sequenced, and sequence is SEQ ID NO: 2.

According to the nucleotide sequence, it is known that the MYB1-1 protein amino acid sequences of the gene code such as SEQ ID Ν Ο:Shown in 1.

The amino acid sequence of MYB1-1 transcription factors： SEQ ID NO: 1

GRSPCCEKA HTNKGAWTKE

EDDRLIAYIR AHGEGCWRSL

PKAAGLLRCG KSCRLRWINY

LRPDLKRGNF TEEEDELI IK

LHSLLGNKWS LIAGRLPGRT

DNEIK YWNT HIRRKLLNRG

IDPATHRPLS EAGVHQDVSS TISFNGVKQE NDK NSPNGF VETDEKKI PA VEERCPDLNL

DLRISPPPYH QTQQHADPFK TGGKTLCLFC SLGVK SKQC ICS IDTAASS SGNNTNTAYD

FLGLKTGFLD YRSLE K*

GhMYB-encoding gene nucleotide sequence： SEQ ID NO:2 1 ATGGGAAGGT CTCCTTGCTG TGAGAAAGCT CATACAAACA AAGGTGCATG GACCAAAGAA

61 GAAGATGATC GCCTCATCGC TTACATCCGA GCCCACGGTG AAGGTTGCTG GCGTTCTCTC

121 CCTAAGGCTG CTGGCCTTCT CCGTTGTGGC AAAAGTTGCA GGCTTCGTTG GATCAACTAC

181 TTAAGACCTG ACCTCAAACG TGGCAACTTC ACTGAGGAAG AAGATGAACT CATTATCAAG

241 CTCCATAGCC TTCTCGGCAA CAAGTGGTCT CTTATAGCCG GGAGATTACC AGGAAGAACG

301 GATAATGAGA TAAAGAATTA CTGGAACACA CACATAAGGA GGAAGCTATT GAACAGAGGA

361 ATCGATCCAG CGACTCACAG GCCACTGAGT GAGGCAGGTG TTCATCAGGA TGTGTCATCC

421 ACAATATCCT TCAATGGTGT TAAACAAGAA AATGACAAGA TGAATAGCCC CAATGGTTTT

481 GTGGAGACAG ATGAGAAGAA AATCCCAGCA GTTGAAGAAA GGTGTCCAGA CTTGAATTTG

541 GACCTAAGAA TCAGCCCACC TCCTTACCAT CAAACCCAGC AGCACGCAGA TCCATTCAAA

601 ACTGGAGGGA AAACCCTCTG TTTATTTTGC AGTTTGGGAG TTAAAAACAG CAAACAATGC

661 ATTTGCAGCA TCGATACTGC TGCCAGCAGC AGTGGCAACA ACACCAATAC TGCCTATGAT

The gene plant expression vector establishments of 721 3 GhMYB of TTCTTAGGCT TGAAAACTGG TTTCTTGGAT TACAGAAGTT TGGAAATGAA ATGA embodiments 1-1

Plant binary expression vector pCAMBIA2300 is selected (to be purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）As plant expression vector, the 35S promoters that Ρ Τ Π genes contain double enhancers are replaced with Pnos promoters, to reduce expression of the Ρ Τ Π albumen in plant.Select inducible promoter rd29A and terminator

Tnos as GhMYBl-l genes promoter and terminator.

With primer SEQ ID NO:9 and SEQ ID NO:10 (are purchased from ocean Science and Technology Ltd. of Beijing China with plant expression vector pBI121）For template amplification Pnos, using TaKaRa PrimeSTAR HS DNA polymerases.50 μ PCR reaction systems：10 μ 5 xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ PBI121,1.0 ^ PrimeSTAR, 10 μ Μ primer SEQ ID Ν Ο:9 and SEQ ID NO:10 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 56 °C of annealing 30 s, 72 °C of 30 s of extension, after 33 circulations, 72 °C of 10 min of extension.Illustrate that (Promega, T4 connect enzyme reagent kit) is connected to pCAMBIA2300 and obtains pCAMBIA2300-l by kit as PCR product of EcoRI, Bglll digestion by obtained by.

SEQ ID NO: 9 ：

GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 10：

ATCCAGATC AGATCCGGTGCAGATTATTTG

With primer SEQ ID NO:11 and SEQ ID NO:12 using pBI121 as template amplification Tnos, uses

TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 μ 5 xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ pBI121,1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO: 11 and SEQ ID NO:12 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 30 s of extension, after 33 circulations, 72 °C of 10 min of extension.Illustrate that (Promega, T4 connect enzyme reagent kit) is connected to pCAMBIA2300-l and obtains pCAMBIA2300-2 by kit as PCR primer of Sacl, EcoRI digestion by obtained by.

SEQ ID NO: 11：

AAGGAGCTCGAATTTCCCCGATCGTTCAAA

SEQ ID NO: 12：

TCAGAATTCCCAGTGAATTCCCGATCTAGTA

With primer SEQ ID NO:13 and SEQ ID NO:14 with arabidopsis（Colombia's type, purchased from the arabidopsis Biological Resource Center of Ohio State Univ-Columbus USA（Www.arabidopsis.org)) DNA is template amplification arabidopsis rd29A promoters（With reference to Zeng I, et L. 2002, Preparation of total DNA from " recalcit rant plant taxa ", Acta Bot. Sin., 44 (6):Method in 694-697 extracts arabidopsis DNA)₀Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ arabidopsis DNA, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:13 and SEQ ID NO:14 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 30 s of extension, after 33 circulations, 72 °C of 10 min of extension.It is connected to by HindIII, Pstl digestion（Connection method is ibid）PCAMBIA2300-2 obtains pCAMBIA2300-3.

SEQ ID NO: 13：

ACTAAGCTTCCTTCTTGACATCATTCAATTTTA

SEQ ID NO: 14：

TGACTGCAGTCCAAAGATTTTTTTCTTTCCAATAG

With primer SEQ ID NO:15 and SEQ ID NO:16 amplification GhMYBW (template is that embodiment 2 obtains G/z ZBl-l), using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：10 ^ 5xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ GhMYBl-l-pGEM, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:15 and SEQ ID NO:16 each 2.0 μ 1, and

31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, after 33 circulations, 72 °C of 10 min of extension.It is connected to by Pstl, Sacl digestion（Connection method is ibid）PCAMBIA2300-3, obtains plant expression vector rd29A- GhMYB 1-1-2300。

SEQ ID NO: 15：

TGACTGCAGATGGGAAGGTCTCCTTGCTGTG SEQ ID NO: 16：

The rd29A- GhMYB 1-1-2300 expression vectors of AAGGAGCT TCATTTCATTTCCAAACTTCTG embodiments 4 convert Agrobacterium

It is prepared by Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competence：Agrobacterium LBA4404 was drawn to single spot inoculation on the LB solid mediums containing 50 g/ml rifampins and 50 g/ml streptomysins in 1-2 days in advance, 28 °C are cultivated 1 to 2 day.Picking single bacterium colony is inoculated in 5 ml containing 50 μ_§The rifampins of/ι η 1 and 50 μ_§In the LB fluid nutrient mediums of the streptomysins of/ι η 1, under 28 °C shake overnight incubation (；About 12-16 hours) to OD600 values be 0.4, formed seed bacterium solution.Take the bacterium solution after 5 ml culture activation（1 :20 ratio）It is inoculated in LB fluid nutrient mediums of 100 ml containing 50 g/ml rifampins and 50 g/ml streptomysins, 28 °C of shakes cultivate 2-2.5 hours to OD_6(K)=0.8.The min of ice bath bacterium solution 10, shakes up once every 3 min, makes bacterium even into resting state.10 min are centrifuged in 4000 g under 4 °C, supernatant is abandoned；Add certain glycerine of head for precooling 10%（Volume ratio）Resuspension thalline, 4000 g centrifuge 10 min under 4 °C, collect precipitation；Use 10% glycerine（Volume ratio）Repetition is washed 3-4 times；Add 10% glycerine of appropriate ice precooling（Volume ratio）Again suspended bacterial is precipitated, that is, LBA4404 competent cells are made, is dispensed, is saved backup in -70 °C with 40 μ/pipe.

Convert Agrobacterium：Melt competent cell on ice, the rd29A- GhMYB 1-1-2300 plasmids of 1 μ embodiments 3 acquisition, the min of ice bath about 10 after mixing are added into 40 μ competent cell.Competence and DNA mixture are transferred in the electric shock of precooling cup with micropipettor, rapping makes suspension reach bottom, has been careful not to bubble.To be shocked by electricity cup（Purchased from bio-rad) it is put on the slideway of electroporation chamber, promote slideway to put electric shock cup to electroporation chamber base electrode.Using 0.1cm electric shock cup when, MicroPulser (be purchased from Bio-Rad) program is set to " Agr ", and electric shock is once.Electric shock cup is taken out immediately, adds the LB culture mediums of 28 °C of preheatings.It is quickly soft to be beaten competent cell with micropipettor.Suspension is transferred to 1.5 ml centrifuge tube, at 28 °C, cultivated 1 hour with 225 rpm.Take 100 200 μ bacterium solution be coated with corresponding resistance screening culture medium flat plate（LB solid mediums, containing 50 μ_§The rifampins of/ι η 1,50 g/ml streptomysins, 50 μ_§The kanamycins of/ι η 1）, 28 °C of cultures. Embodiment 5 obtains transgene tobacco using agriculture bacillus mediated leaf disc transformation method

With 75% alcohol-pickled tobacco seed（Countries tobacco mid-term storehouse, obtains unit：Tobacco institute of the Chinese Academy of Agricultural Sciences, storehouse numbering I5A00660) 30 s, are washed twice with sterilizing distilled water.8 min are soaked with 0.1% mercuric chloride again, is washed with sterilizing distilled water twice, completes surface sterilizing.The tobacco seed of surface sterilizing is placed in MS (18.78 mM KN0₃, 1.25 mM KH₂P0₄, 20.6 mM H4NO3,1.5 mM MgS0₄, 3.0 mM CaCl₂, 50 μ Μ Κ Ι, 100 μ Μ Η₃ΒΟ₃, 100 M MnSO₄, 30 M ZnSO₄, 1 μ Μ Ν α₂Μο0₄, 0.1 M CoCl₂, 100 μ Μ Ν α₂ΕϋΤΑ, 100 M FeSO₄, 7.4 g/L agar, the g/L of sucrose 30) under aseptic condition germinate, prepare aseptic seedling.Take tests for sterility to be cut into the leaf dish of 5 mmx5 mm sizes, with the min of During Agrobacterium leaf dish 10 of the 1-1-2300 of rd29A-GhMYB containing expression vector in exponential phase, blot bacterium solution, co-cultured 2 days under dark condition（MS culture mediums）.Blade is gone into differential medium（The MS+1 mg/L basic elements of cell division（BA)+0.1 mg/L methyl α-naphthyl acetates（NAA) the mg/L cephalosporins of+50 mg/L kanamycins+500）On, illumination condition (refers to 16 hours illumination/8 hour dark（The Lx of light intensity 3000-4000)) under cultivate 45 days or so, cut after bud is grown up and be transferred to root media（The mg/L cephalosporins of MS+50 mg/L kanamycins+500）Middle culture 30 days or so, preservation is numbered after being transferred to seedling after well developed root system on the MS culture mediums only added with 500 mg/L cephalosporins.

Take the transgenic tobacco leaf of acquisition, extract DNA (arabidopsis DNA extraction methods in be the same as Example 3）, with SEQ ID NO:7 and SEQ ID NO:8 (50 μ PCR reaction systems：5 μ Ι Ο 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:9 standing grain mouthful SEQ ID NO:10 each 2.0 μ 1, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,94 30 s of denaturation, 55 °C of 30 s of annealing, 72 °C of 2 min of extension, after 33 circulations, 72 °C of 10 min of extension), PCR identifications preserve positive plant and the overexpression GhMYBl-l transgene tobaccos of Tod-ToC^ embodiments 6 T are numbered_QDrought-enduring simulated experiment and Function Identification sterilized vermiculite is impregnated with 1/2MS culture mediums.Tod-ToC^ and control tobacco tissue-cultured seedling are transplanted to vermiculite respectively, 25 °C, optical culture/14 hour light culture circulation in 10 hours, pour a 1/2MS within every 5 days, after strong seedling culture 15 days, carry out drought stress experiment, transgene tobacco, arid 14 days (not the watering) of control tobacco, 25 °C, optical culture/14 hour light culture circulation in 10 hours.T0 shows that adjoining tree is all wilted seriously, and T for the Identification of Drought of transfer-gen plant_QC₂、 T₀C₃ ToC₇ T₀Cio T₀Cii T_QC₁₄Six strains have in totally 29 tobaccos 21 can normal growth, show significant drought tolerance（Referring to Fig. 3, with ToC₂、 ToC₇Exemplified by, T.C₃、 T₀Cio T。C_U、 1 )。₁₄Result and T.C₂、 T。C₇It is similar, it is not shown here）. Embodiment 7 verifies G/z Sl-1 gene expressions on transcriptional level

Control tobacco, drought-enduring not notable transgene tobacco T are taken respectively_QFor plant, drought-enduring notable transgene tobacco To for plant（Upgrowth situation is good）The arid leaf 0.05g of 14 days, total serum IgE is extracted with plant RNA extraction kit (Invitrogen).Total serum IgE is determined in 260 nm and 280 nm absorbance with the ultraviolet specrophotometer U-2001 of HITACHI companies, calculates each RNA concentration.According to Invitrogen reverse transcriptions try method shown in neat LI boxes Superscript III Reverse Transcriptase carry out reverse transcription (.2 ag total serum IgEs be used as template, reverse transcription primer SEQ ID NO: 8).Pass through SEQ ID NO:7 and SEQ ID NO:8 amplification GhMYBW, detect MYBl-1 albumen relative expression's situations.Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of reverse transcription.50 μ PCR reaction systems：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 2.0 μ cDNA, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:7 and SEQ ID NO:8 each 2.0 μ 1, and 30 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 55 °C of annealing 30 s, 72 °C of extension lmin, after 29 circulations, 72 °C of 10 min of extension.Product electrophoresis result is as shown in Figure 4：M is DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Bioisystech Co., Ltd）, 1-5 is control tobacco, and 6-18 is drought-enduring notable transgene tobacco T_QFor plant, 19-24 is drought-enduring not notable transgene tobacco T_QFor plant.Stripe size shown in figure and GhMYB- it is in the same size（About 800bp).As a result show there is no foreign gene GhMYBl-l mRNA, drought-enduring notable transgene tobacco T in control tobacco_QTranscription for foreign gene GhMYBl-l in plant is stronger, drought-enduring not notable transgene tobacco T_QTranscription for G Bl-1 in plant is very weak or does not transcribe.

Claims

Claims

1. a MYB class transcription factor for cotton, its amino acid sequence such as SEQ ID NO:Shown in 1.

2. encoding the gene of MYB class transcription factors described in claim 1, its nucleotides sequence is classified as 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 recombinant expression carrier described in claim 3, it is the rd29A-GhMYB 1-1-2300 carriers shown in accompanying drawing 2.

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

6. a kind of method of improvement drought resistance in plants, including：Recombinant expression carrier described in gene or claim 3 or 4 described in claim 2 is imported into plant or plant tissue and makes the gene expression;Preferably, the plant is tobacco.

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

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

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

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