WO2014101121A1 - 一种棉花锌指蛋白（Czf7）及其编码基因与应用 - Google Patents

一种棉花锌指蛋白（Czf7）及其编码基因与应用 Download PDF

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WO2014101121A1
WO2014101121A1 PCT/CN2012/087866 CN2012087866W WO2014101121A1 WO 2014101121 A1 WO2014101121 A1 WO 2014101121A1 CN 2012087866 W CN2012087866 W CN 2012087866W WO 2014101121 A1 WO2014101121 A1 WO 2014101121A1
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plant
seq
gene
expression vector
ghczf7
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PCT/CN2012/087866
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French (fr)
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崔洪志
王建胜
梁远金
刘捷
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创世纪转基因技术有限公司
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Priority to PCT/CN2012/087866 priority Critical patent/WO2014101121A1/zh
Priority to CN201280078044.5A priority patent/CN104884620A/zh
Publication of WO2014101121A1 publication Critical patent/WO2014101121A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the present invention relates to plant proteins and their coding genes and applications, and more particularly to a cotton-derived zinc finger protein (Czf7) and a gene encoding the same, and its use in breeding transgenic plants having improved drought resistance.
  • Czf7 cotton-derived zinc finger protein
  • the impact of drought on crop yields ranks first in many natural adversities, and its harm is equivalent to the sum of other disasters. Many regions are the bottleneck of agricultural development.
  • the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid regions account for 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares.
  • Cubic meters due to lack of water, less than 350-40 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the most severe areas in China, and spring droughts frequently reach 10 years.
  • genes and their expression products can be divided into three categories: (1) genes and products involved in signal cascade amplification systems and transcriptional control; (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) Proteins associated with the uptake and transport of water and ions.
  • genes and products involved in signal cascade amplification systems and transcriptional control (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) Proteins associated with the uptake and transport of water and ions.
  • the system has a further understanding (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.
  • the first aspect of the invention provides a coding gene for a zinc finger protein Czf7 of cotton (this article is named
  • GhCzf7 the sequence of which is SEQ ID NO: 2.
  • a second aspect of the invention provides a recombinant expression vector comprising the gene of the first aspect of the invention, and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, The vector is the rd29A-GhCzf7-2300 vector shown in Fig. 2.
  • a third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
  • a fourth aspect of the present invention provides a method for improving drought resistance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and expressing the gene;
  • the plant is tobacco.
  • a fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant
  • the plant is tobacco.
  • a sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought resistance of a plant and for use in plant breeding Use;
  • the plant is tobacco.
  • the seventh aspect of the present invention provides the gene-encoded protein of the first aspect of the present invention, which has an amino acid sequence as shown in SEQ ID NO: 1.
  • FIG. 1 is a construction flow of a plant expression vector Crd29A-GhCzf7-2300 of GhCzf7;
  • Figure 2 is a plasmid map of the plant expression vector Crd29A-GhCzf7-2300 of GhCzf7.
  • Figure 3 shows the drought resistance of control tobacco and transgenic tobacco (Fig. 3a before drought, Fig. 3b after drought); CK (left): control tobacco; and T1B3 (right): transgenic tobacco lines.
  • Figure 4 shows the results of verification of transcriptional levels of drought-tolerant T1 transgenic tobacco plants and drought-tolerant control tobacco plants.
  • M is Marker (DL2000)
  • 1-8 is a drought-tolerant T1 transgenic tobacco plant
  • 10-13 is a drought-tolerant control tobacco plant
  • 9 is a plasmid positive control.
  • BEST MODE FOR CARRYING OUT THE INVENTION The following examples are provided to facilitate a better understanding of the present invention by those skilled in the art. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.
  • Example 1 Cotton SSH library construction under drought stress:
  • African cotton (National Cotton Medium-Term Library, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) was planted on sterilized vermiculite at 25 °C, photoperiod 16h light/8h dark (light intensity 2000-3000 Lx) Under the conditions of culture, 1/2MS medium (containing 9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM NH4NO3, 0.75 mM MgS0 4 , 1.5 mM CaCl 2 , 50 ⁇ M KI, 100 ⁇ MH 3 ) B0 3 , 100 ⁇ M MnS0 4 , 30 ⁇ M ZnS0 4 , 1 ⁇ M Na 2 MoO 4 , 0.1 ⁇ M CoCl 2 , 100 ⁇ M Na 2 EDTA, 100 ⁇ M FeS0 4 ) once. It was used for experiments when the seedlings were as high as 25-30 cm.
  • test seedlings were divided into two groups, each with 4 pots and 1 pot per pot.
  • the first group is the control group, at 25 °C, photoperiod
  • 16h light / 8h dark (light intensity 2000-3000 Lx) culture normal watering.
  • the second group was the drought treatment group, cultured at 25 °C, photoperiod 16h light/8h dark (light intensity 2000-3000 Lx), stopped watering, treated for 10 days, and cut the top of the two seedlings in time after treatment.
  • the leaves of 3 were quickly frozen in liquid nitrogen and stored in a -70 ° C refrigerator.
  • RNA extraction kit purchased from Invitrogen
  • the absorbance of total RNA at 260 nm and 280 nm was determined by HITACHI's UV spectrophotometer U-2001.
  • the OD 26Q / OD 28Q ratio was 1.8-2.0, indicating a high total RNA purity with 1.0% agarose gel.
  • the total RNA was detected by electrophoresis, and the 28S band was about twice as bright as the 18S band, indicating good RNA integrity.
  • mRNA was isolated using Qiagen's purification of polyA+ RNA from total RNA.
  • Two tester cDNAs with different adaptors were mixed with excess Driver for the first forward subtractive hybridization.
  • the two products of the first forward subtractive hybridization were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment. .
  • the second inhibitory PCR product of the second forward subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) and pGEM-T Easy (purchased according to the procedure of pGEM-T Easy kit)
  • the vector was ligated from the Promega kit.
  • the specific steps are as follows: The following components were sequentially added using a 200 ⁇ l PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 ⁇ 1, 2 ⁇ ⁇ 4 ligase buffer 5 ⁇ l, pGEM-T Easy vector 1 ⁇ l, T4 DNA ligase 1 ⁇ l, ligated overnight at 4 °C.
  • the plates were incubated at 37 ° C for 18 h at LB (ibid) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA) containing 50 ⁇ g/ml ampicillin. Count the number of clear white and blue colonies > 1 mm in diameter in the culture plate and randomly pick 540 white colonies (number: Gh-D2-001 to Gh-D2-540). All white clones were inoculated into 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 ⁇ g/ml ampicillin, cultured overnight at 37 ° C, and glycerol was added to a final concentration of 20%, and stored at -80 ° C. spare.
  • CORNING 96-well cell culture plates
  • the nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-select TM cDNA Subtraction Kit kit) were used to carry out PCR amplification of the bacterial liquid, and 452 positive clones were obtained, and then all the positive clones were sent to the British ⁇ jieji ( Shanghai) Trading Co., Ltd. sequencing.
  • sequence was SEQ ID NO: 3.
  • Sequence analysis indicated that the protein encoded by the sequence belonged to the zinc finger protein.
  • the full-length coding gene corresponding to the sequence of SEQ ID No: 3 was designated as GhCzf7. Its corresponding protein is named Czf7.
  • 1S1 CCA TGT AT CGA ACCACC CAAAA CGAA GTC A AC T&AA CAA TTAAAA CCCTTT ST
  • the first round of PCR amplification was carried out using SEQ ID NO: 5 and the universal primer AAP (provided with the kit), and the cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 4) was used as a template for the first round of PCR amplification.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 l OXEx Buffer, 3 ⁇ 12.5 mM dNTP, 2.0 ⁇ 1 mRNA reverse transcribed cDNA, 1.0 ⁇ 1 Ex Taq (purchased from TAKARA), 10 ⁇ M primer SEQ ID NO: 5 and AAP each with 2.0 ⁇ 1, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50 times with double distilled water, and then 2.0 ⁇ l was used as a template, and the second round of PCR amplification was carried out by using SEQ ID NO: 6 and the universal primer AUAP (provided by the kit).
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 lO X Ex Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ l diluted first round PCR product, 1.0 ⁇ ⁇ ⁇ Taq 10 ⁇ ⁇ primers SEQ ID NO: 6 and AUAP Double distilled water of 2.0 ⁇ 1, and 35 ⁇ ⁇ each.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • SEQ ID NO: 6 and the primer AUAP were subjected to bacterial liquid PCR amplification (the reaction system and the reaction conditions were the same as above), and three positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the cDNA of the gene was obtained. 'End.
  • the resulting 5 ' RACE product was cloned and sequenced, and spliced with the result of SEQ ID NO: 3.
  • the cDNA sequence of GhCZF7 was obtained as SEQ ID NO: 7.
  • the GhCzf7 full-length coding sequence was cloned by SEQ ID NO: 8 and SEQ ID NO: 9.
  • the PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
  • 50 ⁇ l PCR reaction system 10 ⁇ 15 ⁇ PS Buffer, 3 ⁇ 12.5 mM dNTP 2.0 ⁇ 1 cDNA, 1.0 ⁇ 1 PrimeSTAR 10 ⁇ M primers SEQ ID NO: 8 and SEQ ID NO: 9 each 2.0 ⁇ 1, and
  • PCR amplification product plus A tail The PCR product was added 2.5 times of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, the supernatant was removed, air-dried, and dissolved in 21 ⁇ l of double distilled water. Add 2.5 ⁇ l ⁇ Buffer, 0.5 ⁇ 15 mM dATP 1.0 ⁇ 1 Ex Taq. Reaction conditions: reaction at 70 ° C for 30 minutes.
  • a DNA fragment of about 720 bp was recovered (Omega recovery kit), ligated into the pGEM T-easy vector (GhCzf7-pGEM plasmid was obtained, transformed into JM109 method), and 10 white colonies were randomly picked up to contain 50 g/ The mL ampicillin was cultured in LB liquid medium, and cultured at 37 ° C overnight, and then glycerin was added to a final concentration of 20% - 80 ° C for storage.
  • SEQ ID NO: 8 and SEQ ID NO: 9 were subjected to bacterial PCR amplification (reaction system and reaction conditions are the same as above), and three positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the whole of GhCzf7 was obtained.
  • the long coding sequence is SEQ ID NO: 2
  • the amino acid sequence of the encoded protein is SEQ ID NO: 1
  • the plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ⁇ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. .
  • the inducible promoter rd29A and the terminator Tnos were selected as promoters and terminators of the GhCzf7 gene, respectively.
  • Primer SEQ ID NO: 10 and SEQ ID NO: 11 were used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used.
  • PCR reaction system 10 l 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 pBI121 , 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ M primer SEQ ID NO: 10 and P SEQ ID NO: 11 each 2.0 1, 1, and 31 ⁇ ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was digested with EcoRI, Bglll and ligated into pCAMBIA2300 (promega, T4 ligase cassette) to obtain pCAMBIA2300-1.
  • SEQ ID NO: 12 and P SEQ ID NO: 13 Amplification of Tnos using pBI121 as a template, using TAKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was digested with Kpnl, EcoRI and ligated into the pCAMBIA2300-l Cpromega T4 ligase cassette) to obtain pCAMBIA2300-2.
  • PCR reaction system 10 yl 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 Arabidopsis DNA, 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ ⁇ primers SEQ ID NO: 14 and SEQ ID NO: 15 each of 2.0 ⁇ 1, and 31 ⁇ 1 of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the obtained PCR product was digested with HindIII and Sail and ligated to pCAMBIA2300-2 to obtain pCAMBIA2300-3.
  • GhCzf7-pGEM plasmid GhCzf7-pGEM plasmid
  • PrimeSTAR HS DNA polymerase using TaKaRa 50 ⁇ 1 PCR reaction System: 10 ⁇ 1 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 GhCzf7-pGEM plasmid, 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ M primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 ⁇ 1, and 31 ⁇ 1 of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min;), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated (ligation method as above) pCAMBIA2300-3 to obtain plant expression vector rd29A-GhCzf7-2300.
  • Agrobacterium LBA4404 (available from Biovector Science Lab, Inc) Competent preparation: Agrobacterium LBA4404 was plated on LB solid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml streptomycin 1-2 days in advance Single spot inoculation, culture at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml streptomycin, and cultured overnight (about 12-16 h) to OD 6 at 28 °C with shaking. . A value of 0.4 forms a seed broth.
  • Transformation of Agrobacterium Thaw competent cells on ice, add 1 ⁇ ⁇ plasmid to 40 ⁇ of competent cells, mix and ice bath for about 10 min. A mixture of competent cells and rd29A-GhCzf7-2300 plasmid DNA was transferred to an ice-cold electric shock cup with a pipette, and tapped to bring the suspension to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from bio-md) on the slide of the electric shock chamber and push the slide to place the electric shock cup on the base electrode of the electric shock chamber.
  • the program of MicroPulser (purchased from bio-rad) is set to "Agr" and the electric shock is applied once. Immediately remove the electric shock cup and add the pre-warmed LB medium at 28 °C. Quickly and gently spread the cells with a gun. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h. 100-200 ⁇ l of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 ⁇ g/ml rifampicin, 50 ⁇ g/ml streptomycin, 50 ⁇ g/ Ml Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use.
  • Example 5 Obtaining Transgenic Tobacco by Agrobacterium-mediated Transformation
  • the leaves of sterile seedlings were cut into 5 mm ⁇ 5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing expression vector rd29A-GhCzf7-2300 in logarithmic growth phase for 10 min, and the bacterial culture was sucked and co-cultured under dark conditions.
  • Day (MS medium) The leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (NAA) + 50 mg/L kanamycin + 500 mg/L cephalosporin).
  • the cells were cultured for about 45 days under light conditions.
  • the obtained transgenic tobacco leaves were extracted, and DNA (the Arabidopsis thaliana DNA extraction method in Example 3) was used, and SEQ ID NO: 8 and P SEQ ID NO: 9 (50 l PCR reaction system: 5 ⁇ 110 X Ex Buffer, 3 ⁇ 12.5) mM dNTP, 2.0 ⁇ l DNA, 1.0 ⁇ l Ex Taq, 10 ⁇ M primers SEQ ID NO: 8 and P SEQ ID NO: 9 each of 2.0 ⁇ l and 35 ⁇ of double distilled water.
  • DNA the Arabidopsis thaliana DNA extraction method in Example 3
  • SEQ ID NO: 8 and P SEQ ID NO: 9 50 l PCR reaction system: 5 ⁇ 110 X Ex Buffer, 3 ⁇ 12.5) mM dNTP, 2.0 ⁇ l DNA, 1.0 ⁇ l Ex Taq, 10 ⁇ M primers SEQ ID NO: 8 and P SEQ ID NO: 9 each of 2.0 ⁇ l and 35 ⁇ of double distilled water.
  • PCR reaction conditions 94 Pre-denaturation at °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min), PCR identification, numbering of PCR-positive plants (T0B1-T0B20) and save.
  • Example 6 Overexpression of GhCzf7 Transgenic Tobacco T1 Generation Drought Tolerant Simulation Experiment and Functional Identification The sterilized vermiculite was soaked with 1/2 MS medium.
  • T0B1-T0B20 and control tobacco seeds were sown on vermiculite, 15 seeds per pot, 25 ° C, 14 hours light culture/10 hours dark culture cycle, 1/2 MS per week, 25 days after culture, SEQ ID NO: 8 and SEQ ID NO: 9 were subjected to PCR detection to remove negative plants. Drought-tolerant tobacco and control tobacco with the same size were selected for drought-tolerant experiments, and 4-5 seedlings with uniform size were kept in each pot. Transgenic tobacco, control tobacco drought for 14 days (without watering), 25 ° C, 14 hours light culture / 10 hours dark culture cycle.
  • T1 transgenic plants plants grown from T0 transgenic plants
  • T1B3, T1B7, and T1B10 tobaccos showed significant drought resistance
  • Figure 3a and 3b taking T1B3 as an example, the results of T1B7 and T1B10 are similar to those of T1B3, which are not shown here.
  • Example 7 Verification of Czf7 protein expression at the transcriptional level
  • RNA extraction kit (invitrogen) Total RNA extracted. The absorbance values of total RNA at 260 nm and 280 nm were determined by ultraviolet spectrophotometry, and the respective RNA concentrations were calculated. Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription kit Superscript III Reverse Transcriptase (1 ⁇ g of total RNA as a template, reverse transcription primer SEQ ID NO: 9).
  • Czf7 protein was examined by detecting GhCzf7 by SEQ ID NO: 8 and SEQ ID NO: 18.
  • PCR was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and reverse transcribed cDNA as a template.
  • 50 yl PCR reaction system 10 l 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ 1 cDNA, 1.0 ⁇ l PrimeSTAR, 10 ⁇ M primer SEQ ID NO: 8 and P SEQ ID NO: 18 2.0 ⁇ 1, and 30 ⁇ 1 of double distilled water.
  • M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-8 is a drought-tolerant T1 transgenic tobacco plant, and 9 is a plasmid positive control (rd29A-GhCzf7- 2300 plasmid), 10-13 is a drought-tolerant control tobacco plant.
  • DL2000 DNA Ladder Marker
  • 1-8 is a drought-tolerant T1 transgenic tobacco plant
  • 9 is a plasmid positive control (rd29A-GhCzf7- 2300 plasmid)
  • 10-13 is a drought-tolerant control tobacco plant.
  • the strip size shown in the figure is the same as the size of the positive control (approximately 730 bp).
  • the results showed that GhCzf7 transcription was stronger in tobacco plants with drought-tolerant T1 transgenic plants, and GhCzf7 transcription was not resistant to drought-controlled tobacco plants.
  • SEQ ID NO: 18 GGGTGGTATC GATAAAC ATG G

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Abstract

一个来源于棉花的锌指蛋白（Czf7）及其编码基因，以及其在培育抗旱性提高的转基因植物中的应用。

Description

一种棉花锌指蛋白 （Czf7) 及其编码基因与应用
技术领域 本发明涉及植物蛋白及其编码基因与应用， 特别是涉及一个来源于棉花的锌指蛋 白 （Czf7) 及其编码基因， 以及其在培育抗旱性提高的转基因植物中的应用。 背景技术 非生物胁迫,如干旱、盐渍、 极端温度、 化学污染和氧损伤等能够对植物的生长发 育造成严重的危害，对作物产量造成极大损失。其中干旱对作物产量的影响，在诸多自 然逆境中占首位,其危害相当于其它灾害之和，是许多地区是农业发展的瓶颈。据统计， 世界干旱、 半干旱地区占陆地面积的 34%; 我国干旱、 半干旱地区约占国土面积的 52 %,年受旱面积达 200— 270万公顷，全国灌溉区每年缺水约 30亿立方米,因缺水而少 收粮食 350— 400亿公斤； 特别是我国主要产粮区如华北、 东北和西北,是我国缺水最 严重的地区,春旱频繁达到十年九遇。
由于植物的耐胁迫性大多属于数量性状,现有可利用的种质资源匮乏，采用常规育 种技术改良植物胁迫耐性的难度相当大,培育出真正的耐胁迫品种就尤为困难。近年来, 随着对植物抗逆分子机理研究的不断深入和分子生物学技术的迅猛发展， 抗逆研究已 经从生理水平深入到分子水平,促进了植物抗逆基因工程的发展。当植物在受到胁迫时 会产生相应的应答反应,来降低或消除给植株带来的危害。植物的这种应答反应是一个 涉及多基因、 多信号途径、 多基因产物的复杂过程。 这些基因及其表达产物可以分为 3 类： （1 ) 参与信号级联放大系统和转录控制的基因及产物； （2) 直接对保护生物 膜和蛋白质起作用的基因及其表达产物； （3 ) 与水和离子的摄入和转运相关的蛋白 质。 近年来,通过转基因技术提高植物对胁迫耐受能力的研究，以及对胁迫具有耐受能 力的农作物、旱生植物和盐生植物的研究都取得了显著的成果，对胁迫相关基因和信号 转导系统也有了更进一步的了解 (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 l transcriptional activators in abscisic acid signaling. Plant Cell, 15: 63-78. )。
但就目前的研究状况而言,由于其机制十分复杂，许多植物对逆境下的生物化学和 生理学上的响应机制仍有待深入研究。在抗逆应答基因的功能及表达调控方面的研究 将对植物抗逆相关的信号传递途径及信号传递网络系统的研究提供重要的基础。 发明内容 本发明人利用 SSH (抑制差减杂交） 与 RACE ( cDNA末端快速扩增） 相结合的 方法克隆出了棉花的一个锌指蛋白（本文命名为 Czf7)的编码基因， 并测定了其 DNA 序列。 并且发现通过转基因将其导入植株后， 可明显改善转基因植株的抗旱性， 而且 这些性状可稳定遗传。
本发明第一方面提供棉花的一个锌指蛋白 Czf7 的编码基因 （本文命名为
GhCzf7), 其序列为 SEQ ID NO: 2。
本发明第二方面提供一种重组表达载体， 其含有本发明第一方面所述的基因并且 所述基因的核苷酸序列与所述表达载体的表达控制序列可操作地连接； 优选地， 所述 载体为附图 2所示的 rd29A-GhCzf7-2300载体。
本发明第三方面提供一种重组细胞， 其含有本发明第一方面所述的基因或者本发 明第二方面所述的重组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。
本发明第四方面提供一种改善植物抗旱性的方法， 包括： 将本发明第一方面所述 基因或者本发明第二方面所述的重组表达载体导入植物或植物组织并使所述基因表 达； 优选地， 所述植物是烟草。
本发明第五方面提供一种制备转基因植物的方法， 包括： 在有效产生植物的条件 下培养含有本发明第一方面所述基因或者本发明第二方面所述的重组表达载体的植 物或植物组织； 优选地， 所述植物是烟草。
本发明第六方面提供本发明第一方面所述的基因、本发明第二方面所述的重组表 达载体或者本发明第三方面所述的重组细胞用于改善植物抗旱性以及用于植物育种 的用途； 优选地， 所述植物是烟草。
本发明第七方面提供本发明第一方面所述的基因编码的蛋白质， 其氨基酸序列如 SEQ ID N0: 1所示。 附图说明 图 1是 GhCzf7的植物表达载体 Crd29A-GhCzf7-2300;)的构建流程。 图 2是 GhCzf7的植物表达载体 Crd29A-GhCzf7-2300)的质粒图。
图 3是对照烟草和转基因烟草的抗旱性生长情况 （图 3a为干旱前， 图 3b为干旱 后）； CK (左)： 对照烟草； 和 T1B3 (右）： 转基因烟草株系。
图 4是耐干旱 T1代转基因烟草植株和不耐干旱对照烟草植株在转录水平上的验 证结果。 M为 Marker (DL2000), 1-8为耐干旱 T1代转基因烟草植株， 10-13为不耐 干旱对照烟草植株， 9为质粒阳性对照。 具体实施方式 提供以下实施例， 以方便本领域技术人员更好地理解本发明。 所述实施例仅出于 示例性目的， 并非意在限制本发明的范围。 实施例 1. 干旱胁迫下棉花 SSH文库构建：
具体方法为：
利用 Clontech公司的 PCR-selectTM cDNA Subtraction Kit 所示的方法通过抑制差 减杂交方法构建差减文库。 在实验过程中以干旱处理的棉花幼苗的叶片中提取的 mRNA 作为样本 （tester) , 以未处理的棉花幼苗的叶片中提取的 mRNA 作为对照 ( driver)。 具体步骤简述如下：
( 1 ) 供试材料：
非洲棉 （国家棉花中期库， 获取单位中国棉花研究所， 统一编号： ZM-06838 )播 种到灭过菌的蛭石上， 在 25 °C、 光周期 16h光照 /8h黑暗 （光强 2000— 3000 Lx) 条 件下培养， 每周浇 1/2MS培养基（含有 9.39 mM KN03， 0.625 mM KH2P04， 10.3 mM NH4NO3, 0.75 mM MgS04， 1.5 mM CaCl2， 50 μ M KI， 100 μ M H3B03， 100 μ M MnS04， 30 μ M ZnS04, 1 μ M Na2Mo04, 0.1 μ M CoCl2， 100 μ M Na2EDTA, 100 μ M FeS04) 一次。 当苗株高达 25— 30 cm时用于实验。
( 2) 材料处理：
将供试幼苗分为 2组， 每组 4盆， 每盆 1株。 第一组为对照组， 在 25 °C、 光周期
16h光照 /8h黑暗 （光强 2000— 3000 Lx) 培养， 正常浇灌。 第二组为干旱处理组， 25 °C、 光周期 16h光照 /8h黑暗 （光强 2000— 3000 Lx) 条件下培养， 停止浇灌， 处理 10天， 处理完毕后及时剪取两组幼苗顶端 1/3 的叶片， 用液氮迅速冷冻后， 于 -70°C 冰箱中保存。
( 3 ) 总 RNA提取：
分别取对照组和干旱处理组的棉花叶片 0.5 g， 用植物 RNA提取试剂盒 （购自 invitrogen) 提取棉花叶片的总 RNA。 用 HITACHI公司的紫外分光光度计 U-2001测 定总 RNA在 260 nm和 280 nm的吸光度值， OD26Q/OD28Q比值为 1.8— 2.0，表明总 RNA 纯度较高，用 1.0%的琼脂糖凝胶电泳检测总 RNA的完整性， 28S条带的亮度约为 18S 条带的 2倍， 表明 RNA的完整性良好。 使用 Qiagen公司的 Oligotex mRNA纯化试剂 盒 (purification of polyA+ RNA from total RNA)分离 mRNA。
( 4) 抑制差减杂交：
按 Clontech公司的 PCR-selectTM cDNA Subtraction Kit试剂盒所示的方法进行抑制 差减杂交。先将 Driver mRNA和 Tester mRNA分别反转录， 得到双链 cDNA, 再以 2 μ g Tester cDNA和 2 μ g Driver cDNA作为起始材料进行差减杂交。 在 37°C水浴下分别 将 Tester cDNA和 Driver cDNA用 Rsa I 酶切 1.5 h， 然后将酶切后的 Tester cDNA分 成两等份， 连接上不同的接头， 而 Driver cDNA不连接头。两种连有不同接头的 Tester cDNA分别与过量的 Driver 混合， 进行第一次正向差减杂交。将两种第一次正向差减 杂交的产物混合， 再与新变性的 Driver cDNA进行第二次正向差减杂交,通过两次抑 制性 PCR扩增差异表达的片段，使其得到富集。
( 5 ) cDNA差减文库的构建与初步筛选、 克隆、 鉴定
依照 pGEM-T Easy试剂盒的程序， 将所述第二次正向差减杂交 cDNA片段的第 二次抑制性 PCR产物 （使用 QIAquick PCR Purification Kit纯化， 购自 Qiagen) 与 pGEM-T Easy (购自 Promega试剂盒)载体连接， 其具体步骤如下： 用 200 μ 1 PCR管依 次加入下列成分： 纯化的正向差减杂交 cDNA片段的第二次 PCR产物 3 μ 1， 2 Χ Τ4 连接酶缓冲液 5 μ 1, pGEM-T Easy载体 1 μ 1， T4 DNA连接酶 1 μ 1， 于 4°C连接过 夜。 取 10 μ ΐ连接反应产物， 加入到 100 μ 1感受态大肠杆菌 JM109C购自 TAKARA) 中,冰浴 30 min、 热休克 60秒、 冰浴 2 min,另； ¾ 250 μ 1 LB培养液 （含有 1% Tryptone (购自 OXOID), 0.5% Yeast Extract (购自 OXOID), 1% NaCl (购自国药）） 置 37°C 水浴中，以 225 rpm振荡培养 30 min,取 200 μ 1菌液接种于含 50 μ g/ml氨苄青霉素的 LB (同上） /X-gal/IPTG ( X-gal/IPTG购自 TAKARA) 培养板上, 37°C培育 18 h。 计数 培养板中直径 > 1 mm 的清晰白色及蓝色菌落数， 随机挑取 540 个白色菌落 (编号： Gh-D2-001至 Gh-D2-540)。将所有白色克隆接种于含有 50 μ g/ml氨苄青霉素的 LB 液 体培养基的 96孔细胞培养板 (CORNING)中， 37°C培养过夜后加甘油至终浓度 20%, 于 - 80°C保存备用。以巢式 PCR 引物 Primer 1和 Primer 2R( Clontech公司的 PCR-selectTM cDNA Subtraction Kit试剂盒自带） 进行菌液 PCR扩增， 得到 452个阳性克隆,然后将 所有阳性克隆送英潍捷基 （上海） 贸易有限公司测序。
( 6) 差异克隆的 cDNA测序分析： 将 DNA测序结果去除载体和不明确序列及冗余的 cDNA后， 共得到 405个有效 EST(unigene)。 实施例 2 棉花锌指蛋白编码基因 GhCzf7的克隆
克隆子 Gh-D2-324去掉冗余 DNA后， 序列为 SEQ ID NO: 3， 序列分析表明该序 列编码的蛋白质属于锌指蛋白， 本文将 SEQIDNo: 3序列对应的全长编码基因命名 为 GhCzf7， 其对应的蛋白命名为 Czf7。
SEQ ID O; 3
-、·、一- --^ - -—、： ： ■■· - 《-:.'二 ： ~ -一, ；！ 、
61 CA ATCAGTA ACCG GAT T CGACCTCAAC ¾TGCCGGCTT T¾CCGGAA T C CGCCA^CT
121 ¾A :TTCTSTG TTCTGC GC CGG GATGAT GAASTGCAAA GCCCA.CAC C TGCTAAG¾.AS
1S1 CCA:TGT AT CGA ACCACC CAAAA CGAA GTC A AC T&AA CAA TTAAAA CCCTTT ST
241 AGT' TAGAT AACAA CC¾ TGAA TTTG T GT'T T TT TT'T TGGTQa SAC A T T
301 TCC'TGT GhCZF7全长编码基因的克隆
根据已经获得的 SEQIDNo: 3序列， 设计如下三条特异性引物， 作为 5' RACE 的 3' 端特异性引物。
GhCEFT GSPlr SEQ ID 4：
GhCZF? GSP2 SEQ 10 NO: δ; GhCZF? GSF3; SEQ ID NO: 6: 实验步骤按试剂盒说明书操作 （ 5 ' RACE System for Rapid Amplification of cDNA Ends试剂盒购自 invitrogen公司）。
用 SEQIDNO: 5与通用引物 AAP (试剂盒自带）， 以 mRNA逆转录的 cDNA (反 转录引物 SEQIDNO: 4) 为模板进行第一轮 PCR扩增， 具体步骤如下：
50 lPCR反应体系： 5 μ 1 lOXEx Buffer, 3 μ 12.5 mM的 dNTP， 2.0 μ 1 mRNA 反转录的 cDNA, 1.0 μ 1 Ex Taq (购自 TAKARA)、 10 μ M的引物 SEQ ID NO: 5和 AAP各 2.0 μ 1, 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循 环 （94°C 变性 30s， 55°C退火 30 s， 72 °C 延伸 lmin)， 72 °C 延伸 10 min。 所得的 PCR产物用双蒸水稀释 50倍后取 2.0 μ 1作为模板， 用 SEQ ID NO: 6与 通用引物 AUAP (试剂盒自带） 进行第二轮 PCR扩增， 具体步骤如下：
50 y l PCR反应体系： 5 μ 1 lO X Ex Buffer, 3 μ 1 2.5 mM的 dNTP， 2.0 μ 1稀释 的第一轮 PCR产物， 1.0 μ Ι Εχ Taq 10 μ Μ的引物 SEQ ID NO: 6和 AUAP各 2.0 μ 1， 以及 35 μ ΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环 （ 94°C 变性 30 s, 58 °C退火 30 s， 72 °C 延伸 1 min)， 72 °C 延伸 10 min。 回收第二次 PCR产物中 片段约为 600bp条带（Gel Extraction Kit购自 OMEGA) , 并将其连接于 pGEM-T Easy Vector，然后转化到 JM109 (具体方法同上）， 随机挑取 10个白色菌落于含有 50 μ g/mL 氨苄青霉素的 LB液体培养基中培养， 37°C培养过夜后加甘油至终浓度 20%， -80°C保 存备用。 SEQ ID NO: 6与引物 AUAP进行菌液 PCR扩增（反应体系及反应条件同上）, 得到 3 个阳性克隆,送英潍捷基 （上海） 贸易有限公司测序测序,获得该基因的 cDNA 的 5 ' 端。
所得的 5 ' RACE产物克隆测序后， 与 SEQ ID NO: 3结果拼接。 获得 GhCZF7 的 cDNA序列 SEQ ID NO: 7。
SEQ ID NO: 7：
1 CTTCAAAAAC TTCCACTTTT CTCTATTGTT TGATTATGGC TTTAGAAGCT ATTAACTCTC
61 CAACCTCAGC CACCGCTCCT TTCCGCTTCG ATGATACTAA TTTACACTGC CTTGAATCCT
121 TTACTAAGCG TAAGCGTTCA AAACGACCAC GTCATCTCGA CCATGTTACC GCCGAGGAAG
181 AATATTTAGC TCTGTGTCTT ATCATGCTAG CACGCGGCGG TGGCCAACGA TTTCCATCTC
241 CAACCACCAT GCCGGAACCC ACCTCGACGG AGGAGCAGAA ACCGAGTTAC AAGTGCAGTA
3 01 TCTGTAACAA GGCGTTTAAC TCTTACCAGG CTTTGGGTGG ACATAAAGCC AGCCACCGTA
361 AGCTTTCCGG TGGGAACGAC GATCAGACGA CATCGGCGAC CGGGACAGCC GGTGGGGTTG
421 TTACATCATC CGGCGGGAAG TCACATGAGT GTTCCATCTG CCA AAAAGC TTCCCTACGG
481 GTCAGGCCTT GGGAGGCCAT AAAAGATGCC ACTACGAAGG TGGAACGGGT AATAATAACG
541 CCGGCGCCAC TGCCACCGCT AGCAGCGTGA CGGTATCTGA AGGAGTGGGG TCAACCAACA
601 CAAACACCAA TACCCATATC AGTAACCGGG ATTTCGACCT CAACATGCCG GCTTTACCGG
661 AATTCTCGCC AGCTAATTTC TTTGTTTCTG CCGCCGGTGA TGATGAAGTG CAAAGCCCAC
721 ACCCTGCTAA GAAGCCATGT TTATCGATAC CACCCAAAAT CGAAGTCAAC TAAACAATTT
781 AAAACCCTTT TTTTAGTTTT AGATAACAAT TCCATGAATT TTTGTTGTTT TTTTTTTTTG
841 GTGAGACGAT TTTTTCCTGT 根据 SEQ ID NO: 7 序列设计一对引物如下: GhCzfTFt SEQ ID NO: S::
A GGC T AG¾¾GCTA ^AAC C
GhCzfJRi SEQ ID NO:
通过 SEQ ID NO: 8和 SEQ ID NO: 9来克隆 GhCzf7全长编码序列。
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以棉花的 cDNA为模板进行 PCR 反应。 50 μ 1PCR反应体系： 10 μ 15XPS Buffer, 3 μ 12.5 mM的 dNTP 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR 10 μ M的引物 SEQ ID NO: 8和 SEQ ID NO: 9各 2.0 μ 1， 以及
30 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5min 33个循环 （94°C 变性 30 s 58 °C退火 30 s, 72 °C 延伸 2min 72 °C 延伸 10 min
PCR扩增产物加 A尾： PCR产物加 2.5倍的无水乙醇， -20°C放置 10分钟，离心， 去上清，晾干，用 21 μ 1双蒸水溶解。加入 2.5 μ 1 ΙΟΧΕχ Buffer, 0.5 μ 15 mM的 dATP 1.0 μ 1 Ex Taq。反应条件：70°C反应 30分钟。将得到约 720 bp的 DNA片段回收（Omega 回收试剂盒），连接至 pGEM T-easy载体上（得到 GhCzf7-pGEM质粒），转化 JM109方 法同上)， 随机挑取 10个白色菌落于含有 50 g/mL氨苄青霉素的 LB 液体培养基中 培养， 37°C培养过夜后加甘油至终浓度 20% -80°C保存备用。 SEQ ID NO: 8与 SEQ ID NO: 9进行菌液 PCR 扩增 （反应体系及反应条件同上） ， 得到 3个阳性克隆,送至英 潍捷基 （上海） 贸易有限公司测序， 得到 GhCzf7的全长编码序列为 SEQ ID NO: 2 其编码的蛋白的氨基酸序列为 SEQ ID NO: 1
Czf7蛋白的氨基酸序列： SEQ ID NO: 1
1 MALEAINSPT SATAPFRFDD
21 TNLHCLESFT KRKRSKRPRH
41 LDHVTAEEEY LALCLIMLAR
61 GGGQRFPSPT TMPEPTSTEE
81 QKPSYKCSIC NKAFNSYQAL
101 GGHKASHRKL SGGNDDQTTS
121 ATGTAGGVVT SSGGKSHECS
141 ICHKSFPTGQ ALGGHKRCHY
161 EGGTGNNNAG ATATASSVTV
181 SEGVGSTNTN TNTHISNRDF
201 DLNMPALPEF SPA FFVSAA 22 1 GDDEVQSPHP AKKPCLS I PP
241 KIEVN*
GhCzf7编码基因的核苷酸序列： SEQ ID NO: 2
1 ATGGCTTTAG AAGC A AA CTCTCCAACC TCAGCCACCG CTCCTTTCCG CTTCGATGAT
61 ACTAATTTAC ACTGCCTTGA ATCCTTTACT AAGCGTAAGC GTTCAAAACG ACCACGTCAT
121 CTCGACCATG TTACCGCCGA GGAAGAATAT TTAGCTCTGT GTCTTATCAT GCTAGCACGC
181 GGCGGTGGCC AACGATTTCC ATCTCCAACC ACCATGCCGG AACCCACCTC GACGGAGGAG
241 CAGAAACCGA GTTACAAGTG CAGTATCTGT AACAAGGCGT TTAACTCTTA CCAGGCTTTG
3 01 GGTGGACATA AAGCCAGCCA CCGTAAGCTT TCCGGTGGGA ACGACGATCA GACGACATCG
361 GCGACCGGGA CAGCCGGTGG GGTTGTTACA TCATCCGGCG GGAAGTCACA TGAGTGTTCC
421 ATCTGCCATA AAAGCTTCCC TACGGGTCAG GCCTTGGGAG GCCA AAAAG ATGCCACTAC
481 GAAGGTGGAA CGGGTAATAA TAACGCCGGC GCCACTGCCA CCGCTAGCAG CGTGACGGTA
541 TCTGAAGGAG TGGGGTCAAC CAACACAAAC ACCAA ACCC A A CAGTAA CCGGGATTTC
601 GACCTCAACA TGCCGGCTTT ACCGGAATTC TCGCCAGCTA ATTTCTTTGT TTCTGCCGCC
661 GGTGATGATG AAGTGCAAAG CCCACACCCT GCTAAGAAGC CATGTTTATC GATACCACCC
721 AAAATCGAAG TCAACTAA 实施例 3 GhCzf7基因植物表达载体构建
选择植物双元表达载体 pCAMBIA2300 (购自北京鼎国昌盛生物技术有限责任公 司） 作为植物表达载体， 用 Pnos启动子替换 ΝΡΤΠ基因含双增强子的 35S启动子， 以降低 ΝΡΤΠ蛋白在植物中的表达。 选择诱导型启动子 rd29A及终止子 Tnos分别作 为 GhCzf7基因的启动子和终止子。
用引物 SEQ ID NO: 10和 SEQ ID NO: 11以植物表达载体 pBI121 (购自北京华夏 远洋科技有限公司） 为模板扩增 Pnos， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。
50 y l PCR反应体系： 10 l 5 X PS Buffer, 3 μ 1 2.5 mM的 dNTP， 1.0 μ 1 pBI121 , 1.0 μ 1 PrimeSTAR、 10 μ M的引物 SEQ ID NO: 10禾 P SEQ ID NO: 11各 2.0 μ 1， 以及 31 μ ΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 (94°C 变性 30 s， 56 °C退火 30 s， 72 °C 延伸 30 s)， 72 °C 延伸 10 min。 通过 EcoRI、 Bglll酶切后将所得 PCR产物连接到 pCAMBIA2300 (promega, T4连接酶盒)获得 pCAMBIA2300-l。 SEQ 10 NO: 10
GCACGAATTC GGCGGGAAAC: GACAATCTGA
SEQ 10 NO: l
ATCCAGAICTAGATCCGG1GCAGATTATTTG
SEQ ID NO: 12禾 P SEQ ID NO: 13以 pBI121为模板扩增 Tnos， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ ΐ PCR反应体系： 10 μ 1 5 X PS Buffer, 3 μ 1 2.5 mM 的 dNTP， 1.0 μ 1 ρΒΙ121 , 1.0 μ 1 Prime STAR 10 μ Μ的引物 SEQ ID NO: 12禾口 SEQ ID NO: 13各 2.0 μ 1， 以及 31 μ 1的双蒸水。 PCR反应条件： 94°C预变性 5 min, 33 个循环 (94°C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 30 s)， 72 °C 延伸 10 min。 通过 Kpnl、 EcoRI酶切后将所得 PCR产物连接到 pCAMBIA2300-lCpromega T4 连接酶盒） 获得 pCAMBIA2300-2。
SEQ ID NO: 12:
AA€GGTACCGAATTTCCCCGATCGTTCAAA
SEQ ID NO: 13；
TCAGAATICCCAeTGAATTCCCGATCTAGTA SEQ ID NO: 14 禾 P SEQ ID NO: 15 以拟南芥 （哥伦比亚型， 购自 TARI, www.arabidopsis.org) DNA为模板扩增拟南芥 rd29A启动子 （参考 Zeng J., et L. 2002, Preparation of total DNA from " recalcit rant plant taxa" , Acta Bot. Sin., 44(6): 694-697 中的方法提取拟南芥 DNA)。采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ 1 PCR 反应体系： 10 y l 5 X PS Buffer, 3 μ 1 2.5 mM的 dNTP， 1.0 μ 1 拟南芥 DNA， 1.0 μ 1 PrimeSTAR、 10 μ Μ的引物 SEQ ID NO: 14和 SEQ ID NO: 15各 2.0 μ 1， 以及 31 μ 1 的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 (94 °C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 30 s), 72 °C 延伸 10 min。 通过 HindIII、 Sail酶切后将所得 PCR产 物连接到 （连接方法同上） pCAMBIA2300-2获得 pCAMBIA2300-3。
SEQ 10 ND: 14；
ACTAAGCTTCCTTCITGACATCATTCAATTTTA SEQ 10 NO: 15；
TGAGTCGACTC¾AAGAITTTTTTC:TTTCCAATAG SEQ ID NO: 16和 SEQ ID NO: 17扩增 GhCzf7 (模板是实施例 2所获得的阳性
GhCzf7-pGEM质粒）， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ 1 PCR反应 体系： 10 μ 1 5 X PS Buffer, 3 μ 1 2.5 mM的 dNTP， 1.0 μ 1 GhCzf7-pGEM质粒， 1.0 μ 1 PrimeSTAR、 10 μ M的引物 SEQ ID NO: 16和 SEQ ID NO: 17各 2.0 μ 1， 以及 31 μ 1 的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 (94 °C 变性 30 s， 58°C退火 30 s, 72 °C 延伸 lmin；)， 72 °C 延伸 10 min。 通过 Kpnl、 Sail酶切后将所得 PCR产物 连接到 （连接方法同上） pCAMBIA2300-3， 获得植物表达载体 rd29A- GhCzf7-2300。
SEQ ID MO : 16；
T&AGGTACCATGGA&AAGAMGGGACAGTAT
SEQ ID NO : 17 :
AAGGTCGACTCAAACCAGGCTCTG&GTATAA 实施例 4 rd29A-GhCzf7-2300表达载体转化农杆菌
农杆菌 LBA4404 (购自 Biovector Science Lab,Inc) 感受态制备： 提前 1-2天将农 杆菌 LBA4404在含 50 μ g/ml利福平和 50 μ g/ml链霉素的 LB固体培养基上划单斑接 种， 28 °C培养 1至 2天。 挑取单菌落接种于 5 ml含 50 μ g/ml利福平和 50 μ g/ml链 霉素的 LB液体培养基中， 28 °C下摇动培养过夜 (约 12-16 h)至 OD6。。值为 0.4， 形成种 子菌液。 取 5 ml活化后的菌液 （1 :20的比例） 接种于 100 ml同样浓度抗生素的 LB 液体培养基中， 28 °C摇动培养 2-2.5 h至 OD6QQ=0.8。 冰浴菌液 10 min, 每隔 3 min摇 匀一次， 令细菌均匀进入休眠状态。 于 4°C下 4000 g离心 10 min， 弃上清液； 加入一 定量冰预冷的 10%甘油重悬浮菌体, 4°C下 4000 g离心 10 min， 收集沉淀； 用冰预冷的 10%甘油重复洗 3-4次； 加入适量冰浴预冷的 10%甘油重新悬浮细菌沉淀， 以 40 μ 1/ 管将其分装， 于 -70°C保存备用。
转化农杆菌： 在冰上融化感受态细胞， 往 40 μ ΐ的感受态细胞中加入 1 μ ΐ的质 粒， 混匀后冰浴约 10 min。 将感受态细胞和 rd29A-GhCzf7-2300质粒 DNA的混合物 用移液枪转移到冰预冷的电击杯中， 轻敲使悬浮液到达底部， 注意不要有气泡。 将电 击杯（购自 bio-md)放到电击室的滑道上，推动滑道将电击杯放至电击室基座电极处。 使用 0.1cm规格的电击杯的时候， MicroPulser (购自 bio-rad) 的程序设置为 "Agr" ， 电击一次 。 立即取出电击杯， 加入 28 °C预热的 LB培养基。 快速而轻柔的用枪将细 胞打匀。 将悬浮液转入 1.5 ml的离心管， 28 °C， 225 rpm培养 1 h。 取 100— 200 μ 1 的菌液涂布于相应的抗性筛选培养基平板上 （LB固体培养基， 含 50 μ g/ml利福平、 50 μ g/ml链霉素、 50 μ g/ml卡那霉素）， 28 °C培养。 筛选阳性转化克隆， 并将其菌液 于 -70°C保存备用。 实施例 5 利用农杆菌介导的转化法获得转基因烟草
用 75%酒精浸泡烟草种子 （国家烟草中期库， 获取单位： 中国农科院烟草所， 库 编号 I5A00660) 30 s, 然后用灭菌双蒸水洗两次。 再用 0.1%升汞浸泡 8 min， 然后用 灭菌双蒸水洗两次，完成表面灭菌。将表面灭菌的烟草种子置于 MS( 18.78 mMKN03， 1.25 mMKH2P04， 20.6 mM NH4N03, 1.5 mMMgS04， 3.0 mM CaCl2, 50 μ MKI, 100 μ MH3BO3, 100 μ MMnS04, 30 μ MZnS04, 1 μ MNa2Mo04, 0.1 μ M CoCl2， 100 μ M Na2EDTA, 100 MFeS04, 7.4g/L琼脂， 蔗糖 30 g/L) 上于无菌条件下发芽， 制备无菌苗。 取无菌苗叶片剪成 5 mmX5 mm大小的叶盘， 用处于对数生长期的含表 达载体 rd29A-GhCzf7-2300的农杆菌浸染叶盘 10min， 吸干菌液， 在黑暗条件下共培 养 2天（MS培养基）。将叶片转到分化培养基（MS+1 mg/L细胞分裂素（BA)+0.1 mg/L 萘乙酸 （NAA) +50 mg/L卡那霉素 +500 mg/L头孢霉素） 上， 光照条件下培养 45天 左右，待芽长大后切下转移到生根培养基 (MS+50 mg/L卡那霉素 +500 mg/L头孢霉素） 中培养 30天左右， 待根系发达后将小苗转入仅加有 500 mg/L头孢霉素的 MS培养基 上进行编号保存。
取获得的转基因烟草叶片， 提取 DNA (同实施例 3 中拟南芥 DNA提取方法）， 用 SEQIDNO: 8禾 P SEQ ID NO: 9(50 lPCR反应体系：5 μ 110 X Ex Buffer, 3 μ 12.5 mM的 dNTP， 2.0 μ 1 DNA, 1.0 μ 1 Ex Taq、 10 μ M的引物 SEQ ID NO: 8禾 P SEQ ID NO: 9各 2.0 μ 1, 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循 环 （94°C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 1 min)， 72 °C 延伸 10 min)， PCR鉴 定， 对 PCR阳性植株进行编号 （T0B1-T0B20) 并保存。 实施例 6 过表达 GhCzf7转基因烟草 T1代植株的耐旱模拟实验及功能鉴定 灭过菌的蛭石用 1/2MS培养基浸透。 T0B1-T0B20及对照烟草种子分别播种在蛭 石上， 每盆播种 15颗种子， 25°C、 14小时光培养 /10小时暗培养循环， 每周浇一次 1/2MS,培养 25天之后， SEQ ID NO: 8和 SEQ ID NO: 9做 PCR检测，去除阴性植株。 选取大小一致的转基因烟草及对照烟草做耐旱实验，每盆保留大小较一致的 4-5棵苗。 转基因烟草、对照烟草干旱 14天 (不浇水)， 25°C、 14小时光培养 /10小时暗培养循环。
T1代转基因植株 （T0代转基因植株的种子长成的植株） 的抗旱性鉴定表明， 对照植 株都萎蔫严重， 而 T1B3、 T1B7、 T1B10三个株系烟草表现出明显的抗旱性 （参见图 3a和 3b， 以 T1B3为例， T1B7、 T1B10的结果与 T1B3类似， 在此未示出）。 实施例 7 在转录水平上验证 Czf7蛋白表达
实施 6中抗旱性好的转基因植株随机选取 8棵 （分别属于上述 3个抗旱株系）， 实施 6中对照植株随机选取 4棵， 干旱 14天叶片 0.05 g， 用植物 RNA提取试剂盒 ( invitrogen) 提取的总 RNA。 紫外分光光度测定总 RNA在 260 nm和 280 nm的吸光 度值， 计算各个 RNA 浓度。 依照 invitrogen 反转录试剂盒 Superscript III Reverse Transcriptase所示方法进行反转录（ 1 μ g总 RNA作为模板，反转录引物 SEQ ID NO： 9 )。 通过 SEQ ID NO:8和 SEQ ID NO: 18检测 GhCzf7， 检测 Czf7蛋白相对表达情况。 采 用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以反转录的 cDNA为模板进行 PCR反应。 50 y l PCR反应体系： 10 l 5 X PS Buffer, 3 μ 1 2.5 mM的 dNTP， 2.0 μ 1 cDNA, 1.0 μ l PrimeSTAR、10 μ M的引物 SEQ ID NO: 8禾 P SEQ ID NO: 18各 2.0 μ 1，以及 30 μ 1 的双蒸水。 PCR反应条件： 94°C预变性 5 min， 28个循环 （94°C 变性 30 s， 58°C退火 30 s， 72°C延伸 lmin)， 72°C延伸 10 min。产物电泳结果如图 4所示： M为 DNA Ladder Marker ( DL2000,购自深圳瑞真生物技术有限公司）， 1-8为耐干旱 T1代转基因烟草植 株， 9为质粒阳性对照（rd29A- GhCzf7-2300质粒）， 10-13为不耐干旱对照烟草植株。 图中所示条带大小与正对照的大小一致 （约为 730bp)。 结果表明，耐干旱 T1 代转基 因烟草植株中 GhCzf7转录较强， 不耐干旱对照烟草植株没有 GhCzf7转录。
SEQ ID NO： 18： GGGTGGTATC GATAAAC ATG G

Claims

权 利 要 求 书
1. 棉花的一个锌指蛋白的编码基因， 被命名为 GhCzf7， 其核苷酸序列如 SEQ ID NO: 2所示。
2. 一种重组表达载体，其含有权利要求 1所述的基因并且所述基因的核苷酸序列 与所述表达载体的表达控制序列可操作地连接。
3. 权利要求 2所述的载体， 其为附图 2所示的 rd29A-GhCzf7-2300载体。
4. 一种重组细胞，其含有权利要求 1所述的基因或者权利要求 2或 3所述的重组 表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。
5. 一种改善植物抗旱性的方法， 包括： 将权利要求 1所述的基因或者权利要求 2 或 3所述的重组表达载体导入植物或植物组织并使所述基因表达； 优选地， 所述植物 是烟草。
6. 一种制备转基因植物的方法， 包括： 在有效产生植物的条件下培养含有权利要 求 1所述的基因或者权利要求 2或 3所述的重组表达载体的植物或植物组织。
7. 权利要求 6所述的方法， 其中所述植物是烟草。
8. 权利要求 1所述的基因、权利要求 2或 3所述的重组表达载体或者权利要求 4 所述的重组细胞用于改善植物抗旱性以及用于植物育种的用途。
9. 权利要求 8所述的用途， 其中所述植物是烟草。
10. 权利要求 1所述的基因编码的蛋白， 其氨基酸序列如 SEQ ID NO: 1所示。
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