CN104837999A

CN104837999A - Cotton zinc finger protein (Czf6) and coding gene and use thereof

Info

Publication number: CN104837999A
Application number: CN201280077200.6A
Authority: CN
Inventors: 何云蔚; 陈淼; 梁丽
Original assignee: Genesis Seed Industry Co ltd
Current assignee: Genesis Seed Industry Co ltd
Priority date: 2012-11-30
Filing date: 2012-11-30
Publication date: 2015-08-12
Anticipated expiration: 2032-11-30
Also published as: WO2014082293A1; CN104837999B

Abstract

Provided are a zinc finger protein (Czf6) from cotton and the coding gene thereof, and the use thereof in the incubation of transgenic plants with increased drought resistance.

Description

Cotton zinc finger protein (Czf6) and coding gene and use thereof

One grows cotton zinc finger protein（) and its encoding gene and application Czf6

The present invention relates to vegetable protein and its encoding gene and application, more particularly to one zinc finger protein from cotton for technical field（) and its encoding gene, and its application in the genetically modified plants that drought resistance is improved are cultivated Czf6.Background technology abiotic stress, such as arid, salt marsh, extreme temperature, chemical contamination and oxygen injury can cause serious harm to growing for plant, and extreme loss is caused to crop yield.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.

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 significant achievement is all achieved to the research for coercing crops, xerophyte and halophytes with tolerance, stress-related genes and signal transduction system there has also been and further understand（Liu Q. 1998. Two transcription factors, DREB1 and DREB2, 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； KANG JY. 2002. Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell, 14: 343-357； ABE H. 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 15: 63-78. )。

But for current research situation, because its mechanism is sufficiently complex, many plants still need to be studied to the biochemistry under adverse circumstance and physiological response mechanism.The research that the related signaling pathways of plant stress-resistance and signal are transmitted with network system is provided important basis by 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 zinc finger protein for cotton（Be named as Czf6 herein) encoding gene, and determine its DNA sequence.And it was found that being conducted into by transgenosis after plant, the drought resistance of transfer-gen plant is can obviously improve, and these characters can stablize heredity.

First aspect present invention provides a zinc finger protein Czf6 for cotton encoding gene（It is named as herein

GhCzf6), its sequence is SEQ ID NO: 2.

Second aspect of the present invention provides a kind of recombinant expression carrier, and it contains the gene described in first aspect present invention and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector；Preferably, the carrier is the rd29A-Gh Czf6-2300 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 plant drought resistance, including：Gene described in first aspect present invention or the recombinant expression carrier described in second aspect of the present invention are imported into plant or plant tissue and make the gene expression；Preferably, the plant is tobacco.

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

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

Seventh aspect present invention provides the protein of the gene code described in first aspect present invention, its amino acid sequence such as SEQ ID N0:Shown in 1.Brief description of the drawings Fig. 1 is GhCzf6 plant expression vector Crd29A-GhCzf6-2300;>Structure flow. Fig. 2 is the plasmid figure of GhCzf6 plant expression vector (rd29A-GhCzf6-2300).

Fig. 3 is the drought resistance growing state for compareing tobacco and transgene tobacco；CK (left side)：Compare tobacco；T1F9 (in）It is (right with T1F12）：Transgene tobacco strain.

Fig. 4 is drought-resistant and the results of the not drought-resistant T1 for transgenic tobacco plant on transcriptional level.M is Marker, 1-8 be drought-resistant T1 for transgenic tobacco plant, 9 one 12 be not drought-resistant T1 for transgenic tobacco plant.

Embodiment provides following examples, to facilitate those skilled in the art to more fully understand the present invention.The embodiment is not intended to limit the scope of the present invention merely for exemplary purpose.Cotton SSH library constructions under the drought stress of embodiment 1：

Specific method is：

Utilize the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit builds subtracted library by Subtractive hybridization method.The mRNA extracted in experimentation using in the blade of the cotton seedling of Osmotic treatment is used as sample（), tester the mRNA extracted using in the blade of untreated cotton seedling is used as control (driver).Specific steps are summarized as follows：

(1) material to be tested：

African cotton（National Cotton mid-term storehouse, obtains unit Cotton research institute, Unified number：ZM-06838) it is seeded on sterilized vermiculite, is cultivated under the conditions of 25 °C, photoperiod 16h/8h (Lx of light intensity 2000-3000), 1/2MS culture mediums are poured weekly（Contain 9.39 mMKN0₃, 0.625 mM KH₂P0₄, 10.3 mMNH₄N0₃, 0.75 mMMgSO₄, 1.5 mMCaCl₂, 50 μ Μ Κ Ι, 100 μ Μ Η₃ΒΟ₃, 100 MMnSO₄, 30 μ Μ ZnS0₄, 1 μ Μ Ν α₂Μο0₄, 0.1 μΜ CoCl₂, 100 μΜ Na₂EDTA, 100 MFeSO₄)-secondary.It is used to test when seedling plant height is up to 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, is cultivated under the conditions of 25 °C, photoperiod 16h/8h (Lx of light intensity 2000-3000), normal to pour.Second group is Osmotic treatment group, 25 °C, cultivate under the conditions of photoperiod 16h/8h (Lx of light intensity 2000-3000), stop 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, preserved in-70 °C of refrigerators.

(3) Total RNAs extraction：

Control group and the g of the cotton leaf of Osmotic treatment group 0.5 are taken respectively, use plant RNA extraction kit（It is purchased from Invitrogen the total serum IgE of cotton leaf) is extracted.Total serum IgE is determined in 260 nm and 280 nm absorbance, OD with the ultraviolet specrophotometer U-2001 of HITACHI companies_26Q/OD_28QRatio is 1.8-2.0, shows that total serum IgE purity is higher, detects the integrality of total serum IgE with 1.0% agarose gel electrophoresis, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.MRNA is separated using the Oligotex mRNA purification kits (purification of polyA+ RNA from total RNA) of Qiagen companies.

(4) Subtractive hybridization：

By the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kits carries out Subtractive hybridization.Using Clontech PCR-Select cDNA Subtraction Kits subtractive hybridization kits, Driver mRNA and Tester mRNA are first distinguished into reverse transcription, obtain double-strand cDNA, then the progress subtractive hybridization using 2 Tester cDNA and 2 ig Driver cDNA as parent material.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 respectively, carry out positive subtractive hybridization for the first time.The product of two kinds of first time subtractive hybridization is mixed, then second of positive subtractive is carried out with Fresh denatured Driver cDNA and is hybridized, the fragment of differential expression is expanded by inhibition PCR twice, it is enriched with.

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

According to the program of pGEM-T Easy kits, the positive subtractive is hybridized to second of PCR product of cDNA fragments（Purified using QIAquick PCR Purification Kit, purchased from Qiagen) it is connected with pGEM-T Easy (being purchased from Promega kits) carrier, it is comprised the following steps that：Following ingredients are sequentially added with 200 μ PCR pipes：The positive subtractive of purifying hybridizes the μ 1,2 of second of PCR primer 3 of cDNA fragments><4 ligase buffer solutions of Τ 5 μ 1, pGEM-T Easy carrier 1 μ 1, the μ 1 of T4 DNA ligases 1, are stayed overnight in 4 °C of connections.10 μ coupled reaction products are taken, are added in 100 μ competence e. coli jm109s (being purchased from TAKARA), the min of ice bath 30, heat shock 60 seconds, the min of ice bath 2,250 μ LB nutrient solutions of another power mouthful（Containing 1% Tryptone (being purchased from OXOID), 0.5% Yeast Extract (are purchased from OXOID), and 1% NaCl (is purchased from traditional Chinese medicines））Put in 37 °C of water-baths, with the min of 225 rpm shaken cultivations 30, take 200 μ bacterium solutions to be inoculated in the LB containing 50 g/ml ampicillins (ibid）On/X-gal/IPTG (X-gal/IPTG is purchased from TAKARA) culture plate, 37 °C of 18 h of cultivation.Count diameter in culture plate>1 mm clear white and blue colonies number, random 540 white colonies of picking (numbering：Gh-D2-001 to Gh-D2-540).All white colonies are inoculated in containing 50 μ_§In 96 porocyte culture plates (CORNING) of the LB fluid nutrient mediums of the ampicillins of/ι η 1, 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 the Primer 2R (PCR-select of Clontech companies^TMCDNA Subtraction Kit kits are carried）Bacterium solution PCR amplifications are carried out, 452 positive colonies is obtained, then send 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 result is removed to carrier and indefinite sequence and redundancy, 405 effective EST (unigene) are obtained.The cotton zinc finger protein gene GhCzf6 of embodiment 2 clone

Clone Gh-D2-153 removes after redundant DNA, and sequence is SEQ IDNo:3, sequence analysis shows that the albumen of the sequential coding belongs to zinc finger protein, herein by SEQ ID No:The corresponding total length encoding gene of 3 sequences is named as GhCzf6, and corresponding albumen is named as Czf6

SEy ID No« 3 1 ；— 、

GCC A- A TCC T^AC TTAATCA A AhGA A A^AAA AG AAA CAAA. CT G' TAAGA T^CG CACG T TCA TG T GTCCCCA TAC TA A G GC

The clone of GhCzf6 total length encoding genes

According to the SEQ ID No obtained:3 sequences, design following three specific primers, are used as 5'RACE 3' end primers.

G Csf6 GSP3: SEQ ID D; 4:

~ _, one, ,-,

GhCsf6 GSP-h SEQ ID D; 5：

CaCG '^SA TG GCACGTGCAA C

GbCsf6 GSP5; SEQ ID NO: 6：

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

With SEQ ID NO:5 (kit is carried with 5' universal primers AAP）, with cDNA (the reverse transcription primer SEQ IDNO of mRNA reverse transcriptions:4) amplification of first round PCR is carried out for template, comprised the following steps that：

50 μ PCR reaction systems：The μ Ex Taq of cDNA 1.0 (being purchased from TAKARA), the 10 μ Μ primer SEQ ID NO of the mM of 53 μ of μ Ι Ο Ε χ Buffer 2.5 2.0 μm of RNA reverse transcriptions of dNTP:Each μ distilled waters of 2.0 μ 1 and 35 of 5 and AAP.PCR reaction conditions：94 °C of 5 min of pre-degeneration, 33 circulations（94 °C of 55 °C of 30 s of denaturation, 72 °C of 30 s of annealing extension lmin), 72 °C of 10 min of extension The PCR primer of gained takes 2.0 μ as template after diluting 50 times with distilled water, with SEQ ID NO:8 carry out second with 3' ends primer AUAP takes turns PCR amplifications, comprises the following steps that：

50 μ P UC ri-R reaction systems：The first of the mM of 53 μ of μ Ι Ο Ε χ Buffer 2.5 dNTP, 2.0 μ dilution

U

Take turns PCR primer, 1.0 lExTaq, 10 μ Μ primer SEQ ID NO:Each 2.0 μ 1 and 35 of 6 and AUAP

<

μ distilled waters.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30s, 72 °C of extension l min), 72 °C of 10 min of extension.Reclaim the band that fragment is about 500bp in second of PCR primer（Gel Extraction Kit are purchased from OMEGA), and pGEM-T Easy Vector are coupled with, then

'

JM109 (specific method is ibid) is transformed into, random 10 white colonies of picking are inoculated in containing 50 μ_§In the LB fluid nutrient mediums of the ampicillins of/ι η 1,37 °C were cultivated after V nights that glycerol adding is to final concentration 20%, and -80 °C save backup.With SEQ ID NO:8 carry out bacterium solution PCR amplifications with 3' ends primer AUAP（Reaction system and reaction condition are ibid）, 7 positive colonies are obtained, wherein 4 clones is chosen and delivers to Invitrogen（Shanghai）Trade Co., Ltd's sequencing sequencing, obtains the cDNA of gene 5' ends. 1 '•;

After the 5'RACE product clonings sequencing of gained, by itself and SEQIDNo:3 sequence assemblies.Obtain GhCzf6 full length cDNA sequence SEQ ID No: 7：; it:- ι κ- ) •

：

¹ G A A AA Q A T ·'ί 'ΐ

ί i G A GT T I; ii-

121 TTCCGACTTA

i- i l CG C A C C TCAT TT GT CAA G CAGT

〕

〔:i

Ι ι·:

^!o .· ^J v.-

3ίΙ GCAACC A T C GTG A TG TTGACCGGA

421 G GCGG A T A GTTTT 'ί' ίτ! ^

£ 1

CGTG T CAG G GQ C C A ATT ATC Ί TATAAAA A A

According to SEQ ID NO:7 sequences Design pair of primers are as follows：

GbfefS: SEQ ID NO: 8：

GbCsf6._:SEQ ID D: 9； Pass through SEQ ID NO:8 and SEQ ID NO:9 clone GhCzf6 total length encoding genes.

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of cotton.The Ρ Ο reaction systems of 50 μ 1：The mM of 10 3 μ of the μ 5xPS Buffer 2.5 μ Μ of 2.0 μ cDNA of dNTP, 1.0 μ PrimeSTAR 10 primer SEQ ID NO:8 standing grain P SEQ ID NO:9 each μ distilled waters of 2.0 μ 1 and 30.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of 30 s of denaturation, 58 °C of annealing 30 s 72V extensions lmin), 72 °C of 10 min of extension

Pcr amplification product adds A tails：The absolute ethyl alcohol of 2.5 times of volumes is added in PCR primer, -20 °C are placed 10 minutes, centrifugation is removed supernatant, dried, and is then dissolved with 21 μ distilled waters.Then 2.5 μ Ι Ο χ Ε χ Buffer, the 0.5 μ 5 mM μ Ι Ο Ε χ Taq of dATP 2.5 are added thereto.Reaction condition：70 °C are reacted 30 minutes.Obtained about 540bp DNA fragmentation is reclaimed（Omega QIAquick Gel Extraction Kits）, and be connected on pGEM T-easy carriers, JM109 (method is ibid) is then converted, random 10 white colonies of picking are inoculated in containing 50 μ_§In the LB fluid nutrient mediums of the ampicillins of/ι η 1, glycerol adding is saved backup for 20% -80 °C to final concentration after 37 °C of overnight incubations.With SEQ ID NO:8 and SEQ ID NO:9 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 7 positive colonies are obtained, wherein 4 positive colonies is chosen and delivers to Invitrogen（Shanghai）Trade Co., Ltd is sequenced, and sequence is SEQ ID NO:2, the amino acid sequence of its protein encoded is SEQ ID NO: 1

The amino acid sequence of Czf6 albumen： SEQ ID NO: 1

1 HAEEHKCQAP SGH LCVKHC

21 -:SFL S?¾.TM LCS CYRDF

41 LEEQQQASSA ¾SS∑ST.3PSS

SI SS AvESVSQ VELLTLPSVK

SI GVPL SAVA ISPVTKQKPQ^

101 QQQQQQQQQQ Q:QQQQQPTRC

121 VCRKF 7GL GFKCKCGITF"

141 CGSHRifPESH SC FDFKKIG

Shang β RSSIARAKPV VKAHVSJ *

The nucleotide sequence of GhCzf6 encoding genes： SEQ ID NO: 2

1 ATSGCGGAGG CCAAGCGCCG SAAGGTCAC.C G CTTT TGT TSA .¾s. 5

；

121 C AAA ^ AAC^A A C T TT GCT AAA C TA TCTACCTC T T CT

,

241 GGAGTT C GG G T C CTGT GCG A C ^G CG^J^ ^. GAAA ^CAA

3 1. CAACAQC ¾¾.CAG A CA AQCAQ AA CAAC AA ¾A A ¾AC G Q GGTG

H TG^TTGCA G AA^ GGG A^ GGA CAAGT CAA ^ GATT CGTT

^ Shang i:: AGAG.A GA TG A TGC CAA CCCGT ^T ^AG AG AGA GhT GT GAG TGA The GhCzf6 gene plant expression vector establishments of embodiment 3

Plant binary expression vector pCAMBIA2300 is selected (to be purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）As plant expression vector, the 35S promoter that Ν Ρ Τ Π genes contain double enhancers is replaced with Pnos promoters, to reduce expression of the Ν Ρ Τ Π albumen in plant.The rd29A promoters and terminator Tnos of induction type are selected respectively as the promoter and terminator of GhCzf6 genes.

With primer SEQ ID NO:10 and SEQ ID NO:L l (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.The Ρ Ο reaction systems of 50 μ 1：The xPS Buffer of 10 μ 5, the mM of 3 μ 2.5 dNTP, the μ PrimeSTAR of 1.0 μ ρ Β Ι 121 1.0,10 μ Μ primer SEQ ID NO:10 standing grain Ρ SEQ ID NO:11 each μ of 2.0 μ 1 and 31 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.Connected as PCR primer of EcoRI, Bglll digestion by obtained by（Promega, T4 ligase box）PCAMBIA2300-l is obtained to pCAMBIA2300.

SEQ ID D K) ：

GCACGAATTCAIAC ATGGACGMCGGAT SEQ IB NO : 11：：

ATCCAGATCIAGATCCGGTGCAGATTATTTG primer SEQ ID NO:12 and SEQ ID NO:13 using pBI121 as template amplification Tnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems： 10 μΐ 5 ><PS Buffer, the mM of 3 μ 1 2.5 dNTP, 1.0 μ pBI121,1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:12 standing grain P SEQ ID NO:13 each μ distilled waters of 2.0 μ 1 and 31.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.Connected as PCR primer of Sacl, EcoRI digestion by obtained by（Promega T4 ligase boxes）PCAMBIA2300-2 is obtained to pCAMBIA2300-l.

SEQ; I'D W 1:2：

SEQ; ID O : 13：

ICASAA JTtCCAGTGAAT CCCGATCTAGTA primer SEQ ID NO:14 standing grain P SEQ ID NO:15 with arabidopsis（Colombia's type, purchased from www.arabidopsis.org) DNA be template amplification arabidopsis rd29A promoters（With reference to Zeng J., et al. 2002, Preparation of total DNA from "recalcitrant plant taxa", Acta Bot. Sin., 44(6):Method in 694-697 extracts arabidopsis DNA).Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：The mM of 10 3 μ of μ 5xPS Buffer 2.5 dNTP, 1.0 μ arabidopsis DNA, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:14 standing grain P SEQ ID NO:15 each μ distilled waters of 2.0 μ 1 and 31.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of 30 s of annealing, s), 72 °C extend 10 min for 72 °C of extensions 30.Connected as PCR primer of HindIII, Pstl digestion by obtained by（Connection method is ibid）PCAMBIA2300-3 is obtained to pCAMBIA2300-2.

SEQ ID NO : Ι4τ

ACTAAGGTTCCTTCTTGACATCATTCAATTTIA

TGAi77 .C CCAAAGATTTTTTTCTTICCMTAG primer SEQ ID NO:16 and SEQ ID NO:The full length sequence of 17 amplification GhCzf6 encoding genes（Template is that embodiment 2 obtains GhCzf6 total length encoding genes）, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：The mM of 10 3 μ of μ 5xPS Buffer 2.5 dNTP, 1.0 μ GhCzf6-pGEM, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:16 standing grain P SEQ ID NO:17 each μ distilled waters of 2.0 μ 1 and 31.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min), 72 °C of 10 min of extension.Connected as PCR product of Pstl, Sacl digestion by obtained by（Connection method is ibid）To pCAMBIA2300-3, plant expression vector rd29A- GhCzf6-2300 are obtained.

SEQ ID ) : 16：

SEQ ID ) : 17：

The rd29A- GhCzf6-2300 expression vectors of embodiment 4 convert Agrobacterium

The preparation of Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competent cell：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_§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 OD₆..It is worth for 0.4, formation seed bacterium solution.Take the bacterium solution after 5 ml activation（1 :20 ratio）100 ml are inoculated in containing 50 μ_§The rifampins of/ι η 1 and 50 μ_§In the LB fluid nutrient mediums of the streptomysins of/ι η 1,28 °C are shaken culture 2-2.5 h to OD_6QQ=0.8.Ice bath bacterium solution 10 min, shake up once every 3 min, make the bacterium even into resting state.10 min are centrifuged in 4000 g under 4 °C, supernatant is abandoned；Add 4000 g under 10% glycerine resuspension thalline of a certain amount of ice precooling, 4 °C and centrifuge 10 min, collect precipitation；Ice-cold 10% glycerine repeats to wash 3-4 times；Then adding 10% glycerine of appropriate ice bath precooling, suspended bacterial is precipitated again, is dispensed, is saved backup in -70 °C with 40 μ/pipe.

Convert Agrobacterium：Melt described competent cell on ice, 1 μ plasmid is added into 40 μ competent cell, the min of ice bath about 10 after mixing.Competent cell and DNA mixture are transferred to the electric shock cup of ice precooling with liquid-transfering gun（Purchased from bio-md) in, rapping makes suspension reach electric shock cup bottom, has been careful not to bubble.The electric shock cup is put on the slideway of electroporation chamber, promotes slideway to put electric shock cup to electroporation chamber base electrode.Using 0.1cm specifications 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 cell with liquid-transfering gun.Suspension is transferred to 1.5 ml centrifuge tube, 1 h of culture is shaken in 28 °C of 225 rpm.100-200 μ bacterium solution is taken to be coated on corresponding resistance screening culture medium flat plate（LB solid mediums, containing 50 μ_§The rifampins of/ι η 1,50 g/ml streptomysins, 50 g/ml kanamycins）, 28 °C of cultures.Embodiment 5 obtains transgene tobacco using Agrobacterium-medialed transformation method

With 75% alcohol-pickled tobacco seed（Countries tobacco mid-term storehouse, obtains unit：Tobacco institute of the Chinese Academy of Agricultural Sciences, storehouse numbering I5A00660) 30 s, are then washed twice with sterilizing distilled water.8 min are soaked with 0.1% mercuric chloride again, is then washed with sterilizing distilled water twice, completes surface sterilizing.The tobacco seed of surface sterilizing is placed in MS solid mediums（Contain 18.78 mM KN0₃ 1.25 mM KH₂P0₄、 20.6 mM H₄N0₃ 1.5 mM MgS0₄、 3.0 mM CaCl₂、 50 μΜ ΚΙ、 100 μΜ Η₃ΒΟ₃、 100 M MnSO₄、 30 M ZnSO₄、 1 μΜ Να₂Μο0₄ 0.1 μΜ CoCl₂ 100 μΜ Na₂EDTA 100 μΜ FeS0₄, 7.4 g/1 fine jades month purports, 30 g/l sucrose）On 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 rd29A-GhCzf6-2300 containing expression vector in exponential phase, blot bacterium solution, co-cultured 2 days under dark condition（MS solid mediums）.Blade is gone into differentiation solid medium（The MS+1 mg/1 basic elements of cell division（BA)+0.1 mg/1 methyl α-naphthyl acetates（NAA) the mg/1 cephalosporins of+50 mg/1 kanamycins+500）On, cultivated 45 days or so under the conditions of photoperiod 16h/8h (Lx of light intensity 2000-3000), cut after bud is grown up and be transferred to rooting solid culture medium（The mg/1 cephalosporins of MS+50 mg/1 kanamycins+500）Middle culture 30 days or so, preservation is numbered after being transferred to seedling after well developed root system on the MS solid mediums only added with 500 mg/L cephalosporins.

The blade for the transgenic tobacco plant that clip is obtained, extracts DNA (arabidopsis DNA extraction methods in be the same as Example 3）, with SEQ ID NO:8 and SEQ ID NO:The amplification identification of 9 performing PCRs（50 μ PCR reaction systems：The mM of 53 μ of μ Ι Ο Ε χ Buffer 2.5 dNTP, 2.0 μ DNA, 1.0 μ Ex Taq, 10 μ Μ primer SEQ ID NO:8 standing grain P SEQ ID NO:9 each μ distilled waters of 2.0 μ 1 and 35.PCR reaction conditions：94 °C of min of pre-degeneration 5,33 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 2 min of extension), 72 °C of 10 min of extension), it is T0F1-T0F20 that PCR is accredited as into positive plant numbering.Embodiment 6 is overexpressed drought resisting simulated experiments of the GhCzf6 transgene tobaccos T1 for plant

Sterilized vermiculite is impregnated with 1/2MS culture mediums.T0F1-T0F20 transgene tobaccos and the seed of control tobacco are sowed on vermiculite respectively, and 15 seeds, 25 °C, optical culture/14 hour light culture circulation in 10 hours are sowed per basin.Pour a 1/2MS within every 5 days, after culture 25 days, it is used to arid per 4-5 more consistent seedling of basin reservation size test, transgene tobacco, arid 14 days (not the watering) of control tobacco, 25 °C, optical culture/14 hour light culture circulation in 10 hours.

T1 is for transfer-gen plant（The plant that TO grows up to for the seed of transfer-gen plant）Identification of Drought show that adjoining tree is all wilted seriously, and seven strains of T1F5, T1F7, T1F9, T1F12, T1F15, T1F16, T1F19 have in totally 35 tobaccos 24 can normal growth, show obvious drought resistance（See Fig. 3, by taking T1F9, T1F12 as an example, T1F5, T1F7, T1F15, T1F16, T1F19 result are similar with T1F9, T1F12, are not shown here）.Embodiment 7 verifies Czf6 protein expressions on transcriptional level

In embodiment 6 24 can normal growth under drought condition 1 for transfer-gen plant in randomly select 8, in embodiment 6 11 can not normal growth under drought condition 1 for transfer-gen plant in randomly select 4, each clip arid g of blade 0.05 of 14 days, uses plant RNA extraction kit（Invitrogen total serum IgE) is extracted.Ultraviolet spectrophotometry total serum IgE calculates each RNA concentration in 260 nm and 280 nm absorbance.Method shown in neat Ll boxes Superscript III Reverse Transcriptase, which is tried, according to invitrogen reverse transcriptions carries out reverse transcription（L μ g total serum IgEs are used as template, reverse transcription primer SEQ ID NO: 9).Pass through SEQ ID NO:8 and SEQ ID NO:9 amplification GhCzf6, detect Czf6 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 5 PS Buffer, 3 μ 2.5 mM dNTP, 2.0 μ cDNA, 1.0 μ Prime STAR, 10 μ Μ primer SEQ ID NO:10 standing grain mouthful SEQ ID NO:11 each 2.0 μ 1, and 30 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,29 circulations（94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension lmin), 72 °C of 10 min of extension.Product electrophoresis result is as shown in Figure 4：M is DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Bioisystech Co., Ltd）, 1-8 is normal growth plant, and 9-12 is to be unable to normal growth plant.Stripe size shown in figure and GhCzf6's is in the same size.As a result show that the transcription of GhCzf6 in normal growth plant is stronger, it is impossible to there is no GhCzf6 to transcribe or transcribe very weak in normal growth plant. It will be understood by those skilled in the art that it is suitable for change and adjustment outside specific description, and the present invention includes all this change and adjustment.Inventor requires the right of the modifications and changes fallen into claims which follow scope and spirit.

Claims

Claims

1. the encoding gene of a zinc finger protein for cotton, is named as GhCzf6, its nucleotide sequence such as SEQ ID NO:Shown in 2.

2. a kind of recombinant expression carrier, it contains the gene described in claim 1 and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector.

3. the carrier described in claim 2, it is the rd29A-GhCzf6-2300 carriers shown in accompanying drawing 2.

4. a kind of recombinant cell, it contains the recombinant expression carrier described in gene or Claims 2 or 3 described in claim 1；Preferably, the recombinant cell is restructuring agrobatcerium cell.

5. a kind of method of improvement plant drought resistance, including：Recombinant expression carrier described in gene or claim 2 or 3 described in claim 1 is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is tobacco.

6. a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the gene or Claims 2 or 3 described in claim 1 or plant tissue are cultivated under conditions of plant is effectively produced.

7. the method described in claim 6, wherein the plant is tobacco.

8. the recombinant expression carrier described in gene, Claims 2 or 3 described in claim 1 or the recombinant cell described in claim 4 are used to improve plant drought resistance and the purposes for plant breeding.

9. the purposes described in claim 8, wherein the plant is tobacco.

10. the albumen of the gene code described in claim 1, its sequence such as SEQ ID NO:Shown in 1.