WO2013181832A1 - 棉花avp1蛋白及其编码基因与应用 - Google Patents

棉花avp1蛋白及其编码基因与应用 Download PDF

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WO2013181832A1
WO2013181832A1 PCT/CN2012/076617 CN2012076617W WO2013181832A1 WO 2013181832 A1 WO2013181832 A1 WO 2013181832A1 CN 2012076617 W CN2012076617 W CN 2012076617W WO 2013181832 A1 WO2013181832 A1 WO 2013181832A1
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plant
seq
tobacco
expression vector
nucleotide sequence
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PCT/CN2012/076617
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English (en)
French (fr)
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王建胜
何云蔚
崔洪志
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创世纪转基因技术有限公司
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Priority to PCT/CN2012/076617 priority Critical patent/WO2013181832A1/zh
Priority to CN201280001632.9A priority patent/CN103842376B/zh
Publication of WO2013181832A1 publication Critical patent/WO2013181832A1/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 in particular to a cotton-derived AVP1 protein and its use in breeding transgenic plants with improved drought resistance and salt tolerance.
  • Drought has limited more than 40% of the Earth's land area to crops, posing a serious threat to global agricultural production and food supply. Drought is one of the main environmental constraints on crop yields.
  • the world's saline-alkali soil is large, about 400 million hectares, accounting for one-third of the irrigated farmland.
  • salt accumulation continues due to low rainfall and rapid evaporation; salt content in the coastal areas is increased due to seawater intrusion.
  • China's saline-alkali soil is mainly distributed in the northwest, north China, northeast and coastal areas.
  • soil salinization is becoming more and more serious.
  • excess Na+ in the soil can have a toxic effect on the normal growth and metabolism of the plant. Therefore, how to increase crop yield under saline environment has become a very important issue in agricultural production in China.
  • the stress resistance of plants is a very complex quantitative trait, and its salt tolerance mechanism involves various levels from plants to organs, tissues, physiology and biochemistry to molecules. Plants respond to stresses when they are stressed to reduce or eliminate the damage to plants. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. These genes and their expression products can be divided into three categories: (1) genes and products involved in signal cascade amplification and transcriptional control; (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; Genes and proteins associated with water and ion uptake and transport.
  • tf-PPase tf-pyrophosphatase
  • the inventors cloned the AVP1 gene from cotton and transformed it into tobacco to obtain transgenic tobacco with improved salt tolerance and drought resistance, which has obvious application prospects in the field of improving plant stress resistance.
  • the inventors cloned the DNA sequence of an AVP1 protein encoding gene (designated herein as GhAVPl-2 gene) using a combination of SSH and RACE. It was found that the introduction of the transgenic plants significantly improved the salt tolerance and drought resistance of the transgenic plants, and these traits were stably inherited.
  • a first aspect of the invention provides an AVP1 class protein of cotton having the sequence SEQ ID No: 1.
  • a second aspect of the invention provides a nucleotide sequence encoding the protein of the first aspect of the invention.
  • the nucleotide sequence encoding the protein has the nucleotide sequence shown in SEQ ID NO: 2.
  • a third aspect of the invention provides a recombinant expression vector comprising the nucleotide sequence of the second aspect of the invention, and wherein the nucleotide sequence is operably linked to an expression control sequence of the expression vector.
  • the expression vector is rd29A-GhAVPl-2-2300, as shown in Figure 2.
  • a fourth aspect of the invention provides a recombinant cell comprising the nucleotide sequence of the second aspect of the invention or the recombinant expression vector of the third aspect of the invention.
  • the recombinant cell is a recombinant Agrobacterium cell.
  • a fifth aspect of the invention provides a method for improving salt tolerance and/or drought resistance of a plant, comprising: introducing the nucleotide sequence of the second aspect of the invention or the recombinant expression vector of the third aspect of the invention Plant or plant tissue and expression of the gene.
  • the plant is tobacco.
  • a sixth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a nucleotide sequence comprising the second aspect of the invention or the recombinant expression vector of the third aspect of the invention under conditions effective to produce a plant Plant or plant tissue.
  • the plant is tobacco.
  • a seventh aspect of the invention provides the protein of the first aspect of the invention, the nucleotide sequence of the second aspect of the invention, the recombinant expression vector of the third aspect of the invention or the fourth aspect of the invention
  • Recombinant cells are used to improve plant salt tolerance and/or drought resistance and for use in plant breeding.
  • the plant is tobacco.
  • Figure 2 Plasmid map of plant expression vector rd29A-GhAVPl-2-2300.
  • the AVP1 gene is a tonoplast pyrophosphatase gene.
  • a subtractive library was constructed by inhibition subtractive hybridization using the method shown by Clontech's PCR-selectTM cDNA Subtraction Kit.
  • the mRNA of the leaves of the drought-treated cotton seedlings was used as a tester during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a driver.
  • the specific steps are as follows:
  • ⁇ cotton 14 (National Cotton Medium-Term Library, obtained by the China Cotton Research Institute, Uniform No.: ZM-30270) was planted on vermiculite-killed vermiculite, cultured at 25 ° C, photoperiod 16 h / 8 h, poured 1 per week /2MS medium (9.39 mM KN0 3 , 0.625 mM KH2P0 4 , 10.3 mM MliNOs, 0.75 mM MgS0 4 , 1.5 mM CaCl 2 , 50 ⁇ ⁇ ⁇ , 100 ⁇ MH 3 B0 3 , 100 ⁇ M MnS0 4 , 30 ⁇ M ZnS0 4 , 1 ⁇ MNa 2 Mo0 4 , 0.1 ⁇ M CoCl 2 , 100 ⁇ M Na 2 EDTA, 100 M FeSO 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 was a control group, which was incubated at 25 ° C with light and was normally watered.
  • the second group was the drought treatment group, 25 °C, light culture, stop burning, and treated for 10 days. After the treatment, the leaves of the top 1/3 of the seedlings were cut in time, and then frozen rapidly with liquid nitrogen at -70°. C is stored in the refrigerator.
  • RNA extraction kit (invitrogen) according to the instructions.
  • the absorbance of total RNA at 260 nm and 280 nm was measured by UV spectrophotometer U-2001 of fflTACffl, and the ratio of OD260/OD280 was 1.8-2.0, indicating that the total RNA purity was high.
  • Total RNA was detected by 1.0% agarose gel electrophoresis.
  • the integrity of the 28S strip is about twice that of the 18S strip, indicating good RNA integrity.
  • Use Qiagen's Oligotex mRNA Purification Kit purification of polyA+ RNA
  • the first group the double-stranded cDNA obtained in the control group and the drought group was digested with Rsal, and then subjected to suppression subtractive hybridization
  • the second group the double-stranded cDNA obtained in the control group and the drought group was digested with Haelll, and then subjected to suppression subtractive hybridization.
  • the other steps and methods for suppressing subtractive hybridization were carried out in strict accordance with the method shown in the Clontech PCR-selectTM cDNA Subtraction Kit product specification, and finally the second PCR products of the two sets of forward subtracted hybrid cDNA fragments were combined.
  • the second PCR product of the combined forward subtractive hybridization cDNA fragment (QIAquick PCR Purification Kit purification, purchased from Qiagen) was ligated to the pGEM-T Easy (purchased from Promega kit) vector according to the product specification of the pGEM-T Easy kit.
  • the specific steps are as follows: The following components were sequentially added using a 200 ul PCR tube: 3 ul of the second PCR product of the purified cDNA fragment, 5 ul of T4 ligase buffer, pulM-T Easy vector lul, T4 DNA ligase lul, at 4° C Connect overnight.
  • the reaction product was ligated to ⁇ competent Escherichia coli JM109 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, and 250 ⁇ M ⁇ liquid medium (1% Tryptone was purchased from OXOID, 0.5% Yeast Extract was purchased from OXOID, 1% NaCl was purchased from Sinopharm. In a shaker at 37 °C, 225 r/min was shaken for 30 min, and 200 ⁇ of bacterial solution was planted in 50 ⁇ g/mL ampicillin, 40 ug/mL.
  • X-gak 24 ug/mL IPTG (X-gal/IPTG was purchased from TAKARA) in LB solid medium (1.5% agar was added to LB liquid medium) and cultured at 37 ° C for 18 h. Count the number of clear white and blue colonies with a diameter >1 mm in the culture plate and randomly select 360 white colonies (numbers: Gh-D001 to Gh-D360). 360 white clones were picked in 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 ug/mL ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C. spare.
  • CORNING 96-well cell culture plates
  • Nested PCR primer Primer 1 (Clontech's PCR-selectTM cDNA Subtraction Kit kit) and Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) are used for PCR amplification. 292 positive clones were obtained, and all positive clones were sequenced in Yingjie Jieji (Shanghai) Trading Co., Ltd.
  • Sequence analysis indicates that the encoded amino acid sequence of the sequence belongs to the AVP1 class protein, and the coding gene corresponding to the clone Gh-D156 is designated as GhAVPl-2 gene.
  • GhAVPl-2 GSP1 SEQ ID No: 4:
  • GhAVPl-2 GSP2 SEQ ID No: 5:
  • the experimental procedure was performed according to the kit instructions (3' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • the first round of PCR amplification was carried out using SEQ ID NO: 4 and 3' end bows, AUAP (provided with the kit), and mRNA reverse transcribed cDNA as a template.
  • the specific steps are as follows: Ex Taq purchased from TAKARA, 50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ Ex Taq, 10 ⁇ bow
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, 33 cycles; extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 100-fold with biguanide water, and then 2.0 ⁇ L was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 5 and 3' end bow AUAP, and the specific steps were as follows: 50 ⁇ l ⁇ Reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ first round of diluted PCR product, 1.0 l Ex Taq, 10 ⁇ primer SEQ ID NO: 5 And AUAP each 2.0 ⁇ 1, and 35 ⁇ double distilled water.
  • PCR reaction conditions predenaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, 33 cycles; extension at 72 °C for 10 min.
  • the second PCR product (QIAquick PCR Purification Kit, purchased from Qiagen) was ligated to pGEM-T Easy Vector, transformed into E. coli JM109 (specifically the same as above), and 10 white colonies were randomly picked up to contain 50 ug/mL ampicillin.
  • the penicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and then glycerin was added to a final concentration of 20%, and stored at -80 ° C until use.
  • AUAP for bacterial PCR amplification 50 ⁇ l ⁇ Reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ bacterial solution, 1.0 ⁇ Ex Taq, 10 ⁇
  • the bows of the SEQ ID NO: 5 and AUAP were 2.0 ⁇ l each, and 35 ⁇ of double distilled water.
  • PCR reaction conditions predenaturation at 94 ° C for 5 min; denaturation at 94 ° C for 30 s, annealing at 58 ° C for 30 s, 72 °C extension for 2 min, 33 cycles; 72 °C extension for 10 min), 4 positive clones were obtained and sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the 3' end of the cDNA of the gene was obtained.
  • GhAVPl-2 GSP4 SEQ ID No: 6:
  • GhAVPl-2 GSP5 SEQ ID No: 7:
  • GhAVPl-2 GSP6 SEQ ID No: 8:
  • the experimental procedure was performed according to the kit instructions (5 'RACE System for Rapid Amplification of cDNA Ends kit, purchased from Invitrogen).
  • SEQ ID No: 6 is used as a primer for reverse transcription of mRNA into cDNA.
  • Ffl SEQ ID NO: 7 and 5' universal primer AAP provided with the kit
  • the mRNA reverse transcription cDNA reverse transcription primer SEQ ID NO: 6
  • Ex Taq was purchased from TAKARA, 50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ total RNA reverse transcribed cDNA, 1.0 ⁇ Ex Taq, 10 ⁇ bow
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, 33 cycles; extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 100 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 with SEQ ID NO: 8 and 3' end bow AUAP, and the specific steps were as follows: 50 ⁇ l ⁇ Reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ first round of diluted PCR product, 1.0 ⁇ Ex Taq, 10 ⁇ bow
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; 94V denaturation for 30 s, annealing at 58 °C for 30 s, 72V extension for 2 min, 33 cycles; 72 °C extension for 10 min.
  • the second PCR product QIAquick PCR Purification Kit, purchased from Qiagen
  • Qiagen 3ul was linked to pGEM-T Easy Vector, converted to JM109 (with The body method was the same as above), and 10 white colonies were randomly picked and cultured in LB liquid medium containing 50 ug/mL ampicillin, and cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20%, and stored at -80 ° C for use.
  • AUAP for bacterial PCR amplification 50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ bacterial solution, 1.0 ⁇ Ex Taq, 10 ⁇
  • the bows of the SEQ ID NO: 8 and AUAP were 2.0 ⁇ l each, and 35 ⁇ of double distilled water.
  • PCR reaction conditions predenaturation at 94 ° C for 5 min; denaturation at 94 ° C for 30 s, annealing at 58 ° C for 30 s, 72 °C extension for 2 min, 33 cycles; 72 °C extension for 10 min), 4 positive clones were obtained and sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the 5' end of the cDNA of the gene was obtained.
  • the obtained 5 ' RACE product was cloned and sequenced, and spliced with the 3 ' RACE product sequencing result.
  • the full-length cDNA sequence of GhAVPl-2 was obtained. According to the full-length cDNA sequence of GhAVPl-2, a pair of bows were designed as follows:
  • GhAVPl-2F SEQ ID No: 9:
  • GhAVPl-2R SEQ ID No: 10:
  • the full length of the GhAVPl-2 gene was cloned by SEQ ID No: 9 and SEQ ID No: 10.
  • the PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
  • PCR reaction conditions 94 °C pre-denaturation 5 min; 94 °C denaturation 30 s, 58 °C annealing 30 s, 72 ° C extension 2 min, 33 cycles; 72 ° C extension 10 min.
  • the PCR amplification product was added with A.
  • the above PCR product was added 2.5 times of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, and the supernatant was removed, dried, and dissolved in 21 ⁇ l of double distilled water.
  • Reaction conditions The reaction was carried out at 70 ° C for 30 minutes.
  • a DNA fragment of about 2300 bp was recovered (Omega recovery kit), ligated into pGEM T-easy vector (purchased from Promega kit), transformed into JM109C method as above), and 10 white colonies were randomly picked up to contain 50 ug/mL ampicillin.
  • the penicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and then glycerin was added to a final concentration of 20%, and stored at -80 ° C until use.
  • PCR amplification of SEQ ID NO: 9 and SEQ ID NO: 10 (using PrimeSAR HS DNA polymerase of TaKaRa, PCR reaction was carried out. 50 ⁇ 1 PCR reaction system: ⁇ 5> ⁇ PS Buffer, 3 ⁇ 1 2.5 mM dNTP , 2.0 ⁇ l of bacterial solution, ⁇ . ⁇ PrimeSTAR, 10 ⁇ of the bow
  • SEQ ID No: 9 and SEQ ID No: 10 each 2.0 ⁇ l, 30 ⁇ l of double distilled water.
  • GhAVPl-2 SEQ ID No: 1 1 MGAAMLSEMA TEIWPVCAV
  • Nucleotide sequence of the gene encoding GhAVPl-2 SEQ ID No: 2
  • the plant binary expression vector PCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the NPTII gene containing the double enhancer 35S promoter was replaced with the Pnos promoter to reduce the expression of prion protein in plants. .
  • the inducible promoters rd29A and Tnos were selected as promoters and terminators of the GhAVPl-2 gene. The specific steps are as follows:
  • SEQ ID NO: 11 and SEQ ID NO: 12 were amplified with the plant expression vector PBI 121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ 1 PCR reaction system: 10 ⁇ 1 5 XPS Buffer, 3 ⁇ 1 2 ⁇ 5 mM dNTP, 1. 0 ⁇ 1 PBI121, 1. 0 ⁇ 1 PrimeSTAR, 10 ⁇ ⁇ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2. 0 ⁇ 1, and 31 ⁇ l of double distilled water.
  • PCR reaction conditions predenaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, 33 cycles; extension at 72 °C for 10 min.
  • the PCR product was digested with EcoRI, Bglll (purchased from New England Biolabs) to pCAMBIA2300 to obtain pCAMBIA2300-1, and the ligation reaction conditions were as shown in the pr omega T4 ligase cassette.
  • HS DNA polymerase 50 ⁇ PCR reaction system: 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ PBI121, 1.0 ⁇ PrimeSTAR 10 ⁇ primers SEQ ID NO: 13 and SEQ ID NO: 14 each 2.0 ⁇ l, and 31 ⁇ l of double steam water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, 33 cycles; extension at 72 °C for 10 min.
  • the PCR product was digested with Sacl, EcoRI (purchased from New England Biolabs) and ligated into pCAMBIA2300-1 to obtain pCAMBIA2300-2.
  • the ligation reaction conditions were the steps indicated in the promega T4 ligase kit.
  • SEQ ID NO: 15 and SEQ ID NO: 16 are based on Arabidopsis thaliana (Columbia type, available from www.arabidopsis.org) DNA (see Zeng I, et L. 2002, Preparation of total DNA from "recalcit rant plant taxa" ", Acta Bot. Sin., 44(6): Method 694-697 for extraction of Arabidopsis DNA) to amplify the Arabidopsis thaliana rd29 A promoter. PrimeSTAR HS DNA polymerase from TaKaRa was used.
  • PCR reaction system 10 ⁇ 5> ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ Arabidopsis DNA, 1.0 ⁇ PrimeSTAR 10 ⁇ primers SEQ ID NO: 15 and SEQ ID NO: 16 each 2.0 ⁇ l, And 31 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, 33 cycles; extension at 72 °C for 10 min.
  • the PCR product was digested with HindIII, Sail (purchased from New England Biolabs) to pCAMBIA2300-2 to obtain pCAMBIA2300-3, and the ligation reaction conditions were as indicated by the promega T4 ligase kit.
  • the AVP1-2 gene was amplified by SEQ ID No: 17 and SEQ ID No: 18, (the template is the pGEM T-easy recombinant vector containing the AVP 1-2 gene obtained in Example 2), and TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 ⁇ 1 PCR reaction system: 10 ⁇ 1 5XPS Buffer, 3 ⁇ 1 2 ⁇ 5 mM dNTP, 1. 0 ⁇ 1 AVP1- 2- pGEM, 1. 0 ⁇ 1 PrimeSTAR, 10 ⁇ M primer SEQ ID NO: 17 And SEQ ID NO: 18 each of 2.0 ⁇ l, and 31 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, 33 cycles; extension at 72 °C for 10 min.
  • the PCR product was ligated to PCAMBIA2300-3 by Sall, Sac I (purchased from New England Biolabs) to obtain the plant expression vector rd29A-GhAVPl-2_2300, and the ligation reaction conditions were as shown in the pr omega T4 ligase kit.
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Competent cell preparation: Agrobacterium LBA4404 was plated in LB solid medium (LB liquid containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml streptomycin) in advance l_2d The medium was added with 1.5% agar) and spotted in a single spot, and cultured at 28 ° C for 1 to 2 days. 5,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
  • 5 ⁇ 0D600 0. 8 ⁇ 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The ice bath solution was shaken for 10 minutes every 3 minutes to allow the bacteria to enter the dormant state evenly.
  • Transformation of Agrobacterium The above Agrobacterium tumefaciens LBA4404 competent cells were thawed on ice, and 1 ⁇ l of the expression vector prepared in Example 3 was added to 40 ⁇ l of competent cells to construct rd29A-GhAVPl-2-2300, mixed and iced. Bath for about 10 minutes. Transfer the above mixture to a pre-cooled electric shock cup with a gun and tap to bring the mixture to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from bio-rad, model Micropulser) 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. Using a 0.
  • 75% alcohol National Tobacco Medium Term Bank, obtained from the Tobacco Institute of the Chinese Academy of Agricultural Sciences, bank number I5A00660. Seeds were simmered for 30 seconds and then immersed in 0.1% liters of mercury for 8 minutes for surface disinfection.
  • the sterilized tobacco seeds were placed in MS solid culture 3 ⁇ 4 (18.78 mM KN0 3 , 1.25 mM KH 2 P0 4 , 20.6 mMMliNOs, 1.5 mMMgS0 4 , 3.0 mM CaCl 2 , 50 ⁇ , 100 ⁇ 3 ⁇ 3 , 100 MMnSO 4 , 30 MZnSO 4 , 1 MNa 2 Mo0 4 , 0.1 MCoCl 2 , 100 MNa 2 EDTA, 100 MFeSO 4 , 7.4 g/L agar, sucrose 30 g/L) were sterilely germinated to prepare sterile seedlings.
  • the leaves of the sterile seedlings were cut into 5 mm X 5 mm leaf discs, and the leaf discs of the expression medium containing the expression vector in the logarithmic growth phase of Example 4 were inoculated with lOmin, and the bacterial liquid was absorbed and co-cultured for 2 days in the dark. (MS solid medium); and leaf discs not infected with Agrobacterium as control leaves.
  • the Agrobacterium-infected leaves were transferred to a differentiation medium (MS solid medium + 1 mg/L BA + 0.1 mg/L NAA + 50 mg/L kanamycin + 500 mg/L cephalosporin), and cultured under light conditions.
  • control leaves were transferred to a differentiation medium (MS solid medium + 1 mg / L BA + O.lmg / L NAA + 50 mg / L kanamycin + 500 mg / L cephalosporin), cultured under light conditions 45
  • a differentiation medium MS solid medium + 1 mg / L BA + O.lmg / L NAA + 50 mg / L kanamycin + 500 mg / L cephalosporin
  • rooting medium MS solid medium + 50 mg/L kanamycin + 500 mg/L cephalosporin
  • the Agrobacterium-infected tobacco seedling leaves were extracted, and genomic DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and PCR was performed using primers SEQ ID No: 17 and SEQ ID No: 18 (Ex Taq was purchased from TAKARA, 50 ⁇ PCR reaction).
  • System 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 17 and SEQ ID No: 18 each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions Pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, 33 cycles; extension at 72 °C for 10 min), selection of 20 successfully transformed transgenic tobacco numbers into T 0 M1-T 0 M20.
  • the sterilized vermiculite was soaked in 1/2 MS medium, and the above cultured T Q M1-T Q M20 transgenic tobacco seedlings and control seedlings were transplanted onto the vermiculite, and cultured at 25 ° C for 10 hours light culture / 14 hours dark Culture cycle, 1/2 MS every 5 days. Seeds were obtained after about 60 days.
  • Example 6 Drought-resistant simulation experiment and functional identification of T1 generation overexpressing GhAVPl-2 transgenic tobacco
  • the sterilized vermiculite was soaked in 1/2 MS medium.
  • the T Q transgenic tobacco T Q N1, T 0 N2 T 0 N3 are sown T Q seeds and control tobacco seeds N4 and T 0 N5 on vermiculite, 25 ° C, 10 hour light culture / 14 hour dark culture cycle, Every 5
  • One 1/2MS was poured once a day, and after 25 days of culture, the bottom leaf was taken and the genomic DNA was extracted (the Arabidopsis DNA extraction method in Example 3), using primers SEQ ID NO: 17 and SEQ ID NO: 18.
  • PCR identification (Ex Taq purchased from TAKARA, 50 ⁇ l ⁇ reaction system: 5 ⁇ 10xEx Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, ⁇ . ⁇ ⁇ ⁇ Taq, 10 ⁇ primers SEQ ID NO: 17 and SEQ ID No: 18 each 2.0 ⁇ 1, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, 33 cycles ; 72 °C extension for 10 min), the negative plants were removed (the control tobacco also picked a leaf).
  • the sterilized vermiculite was soaked in 1/2 MS medium.
  • the T Q transgenic tobacco T Q N1, T 0 N2 T 0 N3 are sown T Q seeds and control tobacco seeds N4 and T 0 N5 on vermiculite, 25 ° C, 10 hour light culture / 14 hour dark culture cycle, 1/2 MS was poured every 5 days, and after 25 days of culture, the lowest leaf was taken, and genomic DNA was extracted (the Arabidopsis DNA extraction method in Example 3) using primers SEQ ID NO: 17 and SEQ ID NO: 18 PCR identification (Ex Taq purchased from TAKARA, 50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ 1 ⁇ ⁇ 10 ⁇ primers SEQ ID NO: 17 and SEQ ID No : 18 each 2.0 ⁇ 1, and 35 ⁇ double distilled water.

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Abstract

本发明提供了一种来源于棉花的AVP1蛋白，及其在培育抗旱性、耐盐性提高的转基因植物中的应用。

Description

棉花 AVP1蛋白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 AVP1蛋白， 以 及其在培育抗旱性、 耐盐性提高的转基因植物中的应用。
背景技术
干旱使得超过 40%的地球陆地面积种植农作物受到限制， 对全球农业生产和粮食供应也 构成了严重的威胁， 干旱是对农作物产量的主要环境限制条件之一。
世界上盐碱土的面积很大， 约有 4亿公顷， 占灌溉农田的 1 /3。 在气候干燥的半干旱、 干旱 地区由于降雨量少、 蒸发剧烈， 盐分不断积累； 海滨地区由于海水倒灌造成土壤含盐量增加。 我国盐碱土主要分布于西北、华北、 东北和滨海地区， 随着大棚面积的逐年增长和栽培年代的 推移， 土壤盐渍化日趋严重。 对绝大多数农作物来说， 土壤中过量的 Na+会对植物体的正常的 生长代谢产生毒害作用。因此如何在盐渍环境下提高作物产量就成为我国农业生产中十分重要 的问题。
采用传统的方法选育耐盐、 抗旱的植物品种固然简便可行， 但进展缓慢， 局限性大。 随着 分子生物学技术的发展， 一大批与植物耐盐、抗旱有关的基因相继得到克隆， 植物转基因技术 有了重大突破， 这为有效利用旱地及盐碱地提高作物产量， 治理盐渍化及荒漠化土地提供了新 的思路和方法。
植物的抗逆性是一个十分复杂的数量性状， 其耐盐机制涉及从植株到器官、 组织、 生理 生化直至分子的各个水平。 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 相关的基因及蛋白质。各国的科学家也为此做了大量的工作， 并取得突破性的进展（Park S. 2005. Up- regulation of a tf-pyrophosphatase (tf-PPase) as a strategy to engineer drought-resistant crop plants. Proc. Natl. Acad. Sci. USA. 102 : 18830-18835 ; ABE H. 2003. A rabid op sis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcrip tional activato rs in abscisic acid signaling. Plant Cell, 15 : 63-78； Zhang ZL. 2011. Arabidopsis Floral Initiator SKB1 Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Splicing through Altering Hi stone H4R3 and Small Nuclear
l Ribonucleoprotein LSM4 Methylation. Plant Cell, 23 : 396 - 411)。 通过生物技术手段， 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。
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占多数， 但 抗逆相关的信号传递途径之间的联系以及整个信号传递网络系统的机理还有待进一步研究。 虽然许多研究机构通过现代生物技术， 获得了各类具有一定耐盐、 抗旱等抗逆能力的转基因 植物， 但还未达到产业化的标准。 因此在提高植物抗逆性方面， 还有许多工作需要做。 发明内容
发明人从棉花中克隆 AVP1基因， 转化到烟草中， 获得耐盐性、 抗旱性提高的转基因烟 草， 在提高植物抗逆性研究领域具有明显的应用前景。
本发明人利用 SSH和 RACE相结合的方法克隆出了棉花的一个 AVP1类蛋白的编码基因 (本文命名为 GhAVPl-2基因） 的 DNA序列。 并发现将其导入转基因植株后， 可明显改善转 基因植株的耐盐性和抗旱性， 而且这些性状可稳定遗传。
本发明的第一方面提供棉花的一个 AVP1类蛋白， 其序列为 SEQ ID No: 1。
本发明的第二方面提供编码本发明第一方面所述的蛋白的核苷酸序列。 优选地， 编码所 述蛋白的核苷酸序列具有 SEQ ID NO: 2所示的核苷酸序列。
本发明的第三方面提供一种重组表达载体，其含有本发明第二方面所述的核苷酸序列， 并且所述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。 在另一个实施方案中， 所述表达载体为 rd29A-GhAVPl-2-2300， 如图 2中所示。
本发明的第四方面提供一种重组细胞， 含有本发明第二方面所述的核苷酸序列或者本 发明第三方面所述的重组表达载体。 在一个实施方案中， 所述重组细胞为重组农杆菌细胞。
本发明的第五方面提供一种改善植物耐盐性和 /或抗旱性的方法， 包括： 将本发明第二 方面所述的核苷酸序列或者本发明第三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。 在一个实施方案中， 所述植物是烟草。
本发明的第六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 培养含有本发明第二方面所述的核苷酸序列或者本发明第三方面所述的重组表达载体的植 物或植物组织。 在一个实施方案中， 所述植物是烟草。
本发明的第七方面提供本发明第一方面所述的蛋白、 本发明第二方面所述的核苷酸序 列、 本发明第三方面所述的重组表达载体或本发明第四方面所述的重组细胞用于改善植物 耐盐性和 /或抗旱性以及用于植物育种的用途。 在一个实施方案中， 所述植物是烟草。 附图说明
图 1· 植物表达载体 rd29A- GhAVPl-2-2300构建流程 （图 la-c)。
图 2. 植物表达载体 rd29A- GhAVPl-2-2300的质粒图谱。
图 3. 转基因烟草和对照植物的抗旱性生长情况； 左： 转基因植株（TiNl-3 ); 右： 对照 烟草 9。
图 4. 转基因烟草和对照植物的耐盐性生长情况； 左： 转基因植株 （ N4-12); 右： 对 照烟草 12。 具体实施方式
AVP1基因是液泡膜 焦磷酸酶基因。
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-select™ cDNA Subtraction Kit所示的方法通过抑制消减杂交 方法构建消减文库。在实验过程中以干旱处理的棉花幼苗的叶子的 mRNA作为样本（tester), 以未处理的棉花幼苗的叶子的 mRNA作为对照 （driver)。 具体步骤简述如下：
( 1 )供试材料：
冀棉 14 (国家棉花中期库， 获取单位中国棉花研究所， 统一编号： ZM-30270)播种到 灭过菌的蛭石上，在 25°C、光周期 16h/8h条件下培养，每周浇 1/2MS培养基（9.39 mM KN03， 0.625 mM KH2P04, 10.3 mM MliNOs, 0.75 mM MgS04， 1.5 mM CaCl2, 50 μ Μ ΚΙ, 100 μ M H3B03， 100 μ M MnS04， 30 μ M ZnS04， 1 μ MNa2Mo04， 0.1 μ M CoCl2， 100 μ M Na2EDTA， 100 M FeSO4) 一次。 当苗株高达 25-30cm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、 光照培养， 正 常浇灌。第二组为干旱处理组， 25°C、光照培养， 停止烧灌， 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子 0.5g， 用植物 RNA提取试剂盒 （invitrogen) 依 照说明书提取棉花的总 RNA。 用 fflTACffl公司的紫外分光光度计 U-2001测定总 RNA在 260nm和 280nm的吸光度值， OD260/OD280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)抑制消减杂交：
为了增加获得 EST (imigene)的有效性， 避免基因无酶切位点及所获得序列在非翻译区， 进行了如下抑制消减杂交。 从前一步骤得到的 mRNA根据 Clontech公司的 PCR-selectTM cDNA Subtraction Kit的说明获得 cDNA。 用 Rsal (购自 New England Biolabs )和 Haein (购 自 New England Biolabs)分别对双链 cDNA进行消化， 做两组抑制消减杂交。 第一组： 对照 组、 干旱组获得的双链 cDNA用 Rsal酶切， 然后进行抑制消减杂交； 第二组： 对照组、 干旱 组获得的双链 cDNA用 Haelll酶切， 然后进行抑制消减杂交。 抑制消减杂交的其他步骤及方 法严格按 Clontech公司的 PCR-selectTM cDNA Subtraction Kit产品说明书中所示的方法进 行， 最后合并两组正向消减杂交 cDNA片段的第二次 PCR产物。
(5) cDNA消减文库的构建与初步筛选、 克隆、 鉴定
合并的正向消减杂交 cDNA片段的第二次 PCR产物（ QIAquick PCR Purification Kit纯化， 购自 Qiagen)与 pGEM-T Easy (购自 Promega试剂盒)载体连接， 依照 pGEM-T Easy试剂盒的 产品说明书， 具体步骤如下： 用 200ul PCR管依次加入下列成分： 纯化 cDNA片段的第二次 PCR产物 3ul， T4连接酶缓冲液 5 ul， pGEM-T Easy载体 l ul， T4 DNA连接酶 lul， 于 4°C 连接过夜。取 ΙΟμΙ^连接反应产物，加入到 ΙΟΟμί感受态大肠杆菌 JM109(购自 TAKARA)中， 冰浴 30min、热休克 60s、冰浴 2min，另加 250 μΙ Β液体培养基 ( 1% Tryptone购自 OXOID, 0.5% Yeast Extract购自 OXOID, 1% NaCl购自国药） 置 37°C摇床中， 225 r/min摇菌 30min， 取 200 ^菌液种植于含 50 ug/mL氨苄青霉素、 40 ug/mL X-gak 24 ug/mL IPTG ( X-gal/IPTG 购自 TAKARA) 的 LB固体培养基 (LB液体培养基中加入 1.5%的琼脂）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 360个白色菌落 (编 号： Gh-D001至 Gh-D360)。 将 360个白色克隆挑于含有 50 ug/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弓 I物 Primer 1 (Clontech公司的 PCR-select™ cDNA Subtraction Kit试剂 盒自带） 和 Primer 2R ( Clontech公司的 PCR-select™ cDNA Subtraction Kit试剂盒自带） 进 行菌液 PCR扩增，得到 292个阳性克隆，对所有阳性克隆在送英潍捷基（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多余的 cDNA后，共得到 180个 EST(unigene)。 经 BlastN发现其中 102条 unigene在 GenBank 中有同源序列（同源性 50%以上）， 33条 EST 功能未知或者为假定蛋白， 另有 45条未获得同源匹配， 推测可能是处于 3 '、 5' 末端非翻译 区的较短序列。
实施例 2、 GhAVPl-2基因的克隆
在上述有同源序列的 unigene中， 克隆子 Gh-D156序列： SEQ ID No: 3：
1 ACTACACCAG CAATGCTTAC AGCCCTGTGC AGGATGTTGC CGACTCTTGC AGGACGGGAG
61 CAGCAACCAA TGTTATATTT GGCCTTGCTT TAGGATACAA ATCTGTGATC ATCCCAATTT
121 TTGCCATAGC AATCAGTATT TTTGTTAGTT TTAGCTTTGC TGCTATGTAT GGGATTGCAG
181 TTGCTGCCCT TGGAATGTTG AGCACCATTG CAACTGGGTT GGCTATCGAT GCTTATGGTC
241 CCATCAGTGA CAATGCTGGA GGTATAGCTG AGATGGCTGG TATGAGCCAT CGCATTCGTG
301 AGAGAACTGA TGCTCTTGAT GCTGCTGGAA ACACCACTGC TGCCATTGGA AAGGGATTTG
361 CCATTGGGTC AGCGGCGTTG GTGTCTTTGG CTCTATTTGG TGCCTTTGTG AGCCGTGCTG
421 CTATCACGAC CGTAGATGTC TTGACCCCAA AAGTTTTCAT TGGTTTAATA GTTGGAGCCA
481 TGCTTCCTTA CTGGTTCTCT GCAATGACCA TGAAGAGTGT GGGAAGTGCT GCTTTAAAGA
541 TGGTTGAGGA GGTTCGCAGA CAGTTCAACA CCATTCCCGG TCTCATGGAG GGTCATGCCA
601 AGCCAGATTA TGCTACCTGT GTCAAAATCT CAACTGATGC CTCTATCAAG GAGATGATCC
661 CTCCTGGTGC CCTTGTTATG CTTACTCCCC TCATTGTTGG AACTTTCTTT GGTGTAGAGA
721 CTCTTTCTGG TGTCCTTGCT GGGGCTCTTG TTTCCGGTGT TCAGATTGCA ATTTCTGCAT
781 CCAATACTGG TGGGGCATGG GACAATGCCA AGAAGT
序列分析（核酸序列在 NCBI Blast)表明该序列的编码的氨基酸序列属于 AVP1类蛋白， 本文将克隆子 Gh-D156对应的编码基因命名为 GhAVPl-2基因。
根据已经获得的 GhAVPl-2基因片段， 设计两条特异性引物， 作为 3 ' RACE的 5'端特 异性引物：
GhAVPl-2 GSP1 : SEQ ID No： 4：
ATGTTATATTTGGCCTTGCTTT
GhAVPl-2 GSP2: SEQ ID No： 5：
ACAAATCTGTGATCATCCCAATTT
实验步骤按试剂盒说明书操作（3' RACE System for Rapid Amplification ofcDNAEnds试 剂盒购自 invitrogen公司）。
用 SEQ ID NO: 4与 3'端弓 |物 AUAP (试剂盒自带）， 以 mRNA逆转录的 cDNA为模板 进行第一轮 PCR扩增。具体步骤如下: Ex Taq购自 TAKARA, 50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ mRNA反转录的 cDNA， 1.0 μΐ Ex Taq、 10 μΜ的弓 |物 SEQ ID NO: 4和 AUAP各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 2min， 33个循环； 72°C 延伸 10 min。
所得的 PCR产物用双熘水稀释 100倍后取 2.0 μΐ作为模板， 用 SEQ ID NO: 5与 3'端弓 | 物 AUAP进行第二轮 PCR扩增，具体步骤如下： 50 μ1 ΡΟ 反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ第一轮稀释的 PCR产物， 1.0 l Ex Taq、 10 μΜ的引物 SEQ ID NO: 5 和 AUAP各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s, 72 °C 延伸 2min， 33个循环； 72°C 延伸 10 min。第二次 PCR产物（QIAquick PCR Purification Kit纯化， 购自 Qiagen) 3ul连接于 pGEM-T Easy Vector, 转化到大肠杆菌 JM109(具体方法同上)，随机挑取 10个白色菌落于含有 50 ug/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: 5与 3'端弓 |物 AUAP进行菌液 PCR扩增 （50 μ1 ΡΟ 反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ菌液， 1.0 μΐ Ex Taq、 10 μΜ的弓 |物 SEQ ID NO: 5和 AUAP各 2.0μ1， 以及 35 μΐ的双 蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s, 72 °C 延伸 2min， 33个循环； 72°C 延伸 10 min) , 得到 4个阳性克隆， 送英潍捷基（上海）贸易有限公司测序, 获得该基因的 cDNA的 3'端。
根据已经获得的 GhAVPl-2基因片段， 设计三条特异性引物， 作为 5 ' RACE的 3 '端特 异性引物：
GhAVPl-2 GSP4: SEQ ID No： 6：
TCACAAAGGC ACCAAATAGA GCC
GhAVPl-2 GSP5: SEQ ID No： 7：
CAATGGCAAA TCCCTTTCCA ATG
GhAVPl-2 GSP6: SEQ ID No： 8：
TCCAGCATTG TCACTGATGG GACC
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）。 SEQ ID No: 6作为 mRNA反转录成 cDNA的引物。
ffl SEQ ID NO: 7与 5'通用引物 AAP (试剂盒自带）， 以 mRNA逆转录的 cDNA (反转 录引物 SEQ ID NO: 6)为模板进行第一轮 PCR扩增，具体步骤如下： Ex Taq购自 TAKARA, 50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ总 RNA反转录的 cDNA, 1.0 μΐ Ex Taq、 10 μΜ的弓 |物 SEQ ID NO: 7和 AAP各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR反应 条件： 94°C预变性 5 min; 94 °C 变性 30 s， 55°C退火 30 s, 72 °C 延伸 2min， 33个循环； 72 °C 延伸 10 min。
所得的 PCR产物用双蒸水稀释 100倍后取 2.0 μΐ作为模板， 用 SEQ ID NO: 8与 3'端弓 | 物 AUAP进行第二轮 PCR扩增，具体步骤如下： 50 μ1 ΡΟ 反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ第一轮稀释的 PCR产物， 1.0 μΐ Ex Taq、 10 μΜ的弓 |物 SEQ ID NO: 8 和 AUAP各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min; 94V 变性 30 s， 58°C退火 30 s, 72V 延伸 2min， 33个循环； 72°C 延伸 10 min。第二次 PCR产物（QIAquick PCR Purification Kit纯化， 购自 Qiagen) 3ul连接于 pGEM-T Easy Vector, 转化到 JM109(具 体方法同上),随机挑取 10个白色菌落于含有 50 ug/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 3'端弓 |物 AUAP进 行菌液 PCR扩增 （50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ菌 液， 1.0 μΐ Ex Taq、 10 μΜ的弓 |物 SEQ ID NO: 8和 AUAP各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR 反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s, 72°C 延伸 2min， 33个循环； 72°C 延伸 10 min) , 得到 4个阳性克隆,送英潍捷基 （上海） 贸易有限公司测序,获得该基因 的 cDNA的 5'端。
所得的 5 ' RACE产物克隆测序后， 与 3 ' RACE产物测序结果拼接。 获得 GhAVPl-2 全长 cDNA序列。 根据 GhAVPl-2全长 cDNA序列设计一对弓 |物如下：
GhAVPl-2F: SEQ ID No： 9：
ATGGGGGCGGCGATGTTGTCGGAG
GhAVPl-2R: SEQ ID No: 10：
CTAAAAGATC TTGAAGAGTA GACC ACC
通过 SEQ ID No: 9和 SEQ ID No: 10来克隆 GhAVPl-2基因全长。
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以棉花的 cDNA为模板进行 PCR反应。
50μ1 ΡΟ 反应体系： ΙΟμΙ 5 xPS Buffer, 3μ1 2.5mM的 dNTP， 2.0μ1 cDNA, Ι .ΟμΙ PrimeSTAR, 10μΜ的弓 I物 SEQ ID No: 9和 SEQ ID No: 10各 2.0μ1， 30μ1的双蒸水。 PCR反应条件： 94 °C 预变性 5min; 94°C 变性 30s，58°C退火 30s，72°C 延伸 2min， 33个循环后； 72°C 延伸 10min。
PCR扩增产物加 A。 将上述 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 21μ1双蒸水溶解。加入 2.5ul ΙΟχΕχ Buffer, 0.5ul 5 mM的 dATP， 2.5ul ΙΟχΕχ Taq。反应条件： 70°C反应 30分钟。将得到约 2300bp的 DNA片段回收（Omega回收试剂盒）， 连接至 pGEM T-easy载体 （购自 Promega试剂盒）， 转化 JM109C方法同上)， 随机挑取 10个 白色菌落于含有 50 ug/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 : 9与 SEQ ID NO : 10进行菌液 PCR扩增 （采用 TaKaRa 的 PrimeSTAR HS DNA聚合酶， 菌液进行 PCR反应。 50μ1 PCR反应体系： ΙΟμΙ 5><PS Buffer, 3μ1 2.5mM的 dNTP， 2.0μ1菌液， Ι .ΟμΙ PrimeSTAR, 10μΜ的弓 |物 SEQ ID No: 9和 SEQ ID No: 10各 2.0μ1， 30μ1的双蒸水。 PCR反应条件： 94°C预变性 5min; 94 °C 变性 30s， 58°C退火 30s, 72V 延伸 2min， 33个循环后； 72°C 延伸 10min。）得到 3个阳性克隆，送英潍捷基（上海） 贸易有限公司测序，序列为 SEQ ID NO : 2。 其蛋白表达序列为 SEQ ID NO: 1。
GhAVPl-2的氨基酸序列： SEQ ID No: 1 1 MGAAMLSEMA TEIWPVCAV
21 IGIAFSLVQW VMVSRVKLTS
41 ERHAS SANSS KNGYGDYLIE
61 EEEGINDHSV VTKCADIQNA
81 ISEGATSFLF TEYQYVGIFM
101 IAFAILIFLF LGSVEGFSMK
121 SQPCTYDKEK MCKPALATAI
141 FSTVSFLLGA ITSVLSGFLG
161 MKIATYANAR TTLEARKGVG
181 KAFIVAFRSG AVMGFLLAAN
201 GLLVLYIAIN LFKLYYGDDW
221 EGLFEAITGY GLGGSSMALF
241 GRVGGGIYTK AADVGADLVG
261 KVERNIPEDD PRNPAVIADN
281 VGDNVGDIAG MGSDLFGSYA
301 ESSCAALWA SISSFGINHD
321 FTGMLYPLLI SSVGILVCLI
341 TTLFATDLFE IKWKEIEPA
361 LKKQLIISTI LMTVGIAIVT
381 WIGVPSSFTI YNFGVQKWK
401 NWQLFLCVGV GLWAGLI IGF
421 VTEYYTSNAY SPVQDVADSC
441 RTGAATNVIF GLALGYKSVI
461 IPIFAIAISI FVSFSFAA Y
481 GIAVAALGML STIATGLAID
501 AYGPISDNAG GIAEMAGMSH
521 RIRERTDALD AAGNTTAAIG
541 KGFAIGSAAL VSLALFGAFV
561 SRAAITTVDV LTPKVFIGLI
581 VGAMLPYWFS AMTMKSVGSA
601 ALK VEEVRR QFNTIPGLME
621 GHAKPDYATC VKISTDASIK
641 EMIPPGALVM LTPLIVGTFF
661 GVETLSGVLA GALVSGVQIA
681 ISASNTGGAW DNAKKYIEAG
701 VSEHARTLGP KGSDPHKAAV
721 IGDTVGDPLK DTSGPSLNIL
741 IKLMAVESLV FAPFFATHGG
761 LLFKIF*
GhAVPl-2编码基因的核苷酸序列： SEQ ID No: 2
1 ATGGGGGCGG CGATGTTGTC GGAGATGGCG ACGGAGATCG TGGTGCCAGT GTGCGCCGTG
61 ATTGGAATAG CTTTCTCGTT GGTGCAGTGG GTGATGGTGT CGCGCGTGAA GCTTACCTCC
121 GAGCGCCATG CGTCGTCGGC GAATAGTAGC AAGAATGGTT ACGGTGATTA CTTGATTGAG
181 GAAGAGGAAG GAATCAATGA CCATAGCGTT GTCACCAAGT GCGCTGATAT TCAAAACGCT
241 ATCTCTGAAG GTGCAACATC CTTTCTTTTT ACTGAATATC AGTATGTTGG CATCTTCATG
301 ATTGCTTTTG CTATTCTGAT TTTCCTCTTC CTGGGCTCTG TCGAGGGCTT CAGCATGAAG 361 AGCCAGCCTT GCACCTATGA TAAGGAGAAG ATGTGCAAAC CAGCTCTTGC GACTGCTATC
421 TTCAGTACTG TATCCTTCTT GCTAGGTGCC ATCACTTCAG TTCTTTCTGG ATTCCTTGGA
481 ATGAAAATTG CTACCTATGC CAATGCAAGA ACAACCTTGG AAGCAAGAAA AGGTGTCGGA
541 AAGGCTTTTA TTGTTGCGTT TAGATCTGGT GCAGTAATGG GTTTTCTCCT TGCAGCAAAT
601 GGTTTATTGG TACTTTACAT TGCCATCAAT CTATTCAAGC TGTACTATGG TGATGACTGG
661 GAAGGCCTTT TTGAGGCTAT TACTGGTTAT GGTCTTGGTG GATCCTCTAT GGCACTCTTT
721 GGAAGAGTTG GTGGTGGTAT CTATACAAAG GCTGCTGATG TCGGTGCTGA TCTCGTAGGA
781 AAGGTTGAAA GGAATATTCC AGAAGATGAC CCAAGAAACC CAGCTGTCAT TGCTGATAAT
841 GTTGGTGATA ATGTCGGGGA CATTGCTGGA ATGGGGTCTG ATCTATTTGG CTCATATGCT
901 GAATCTTCCT GTGCTGCTCT TGTTGTAGCT TCTATATCCT CCTTTGGAAT AAACCATGAC
961 TTCACGGGCA TGTTGTATCC TCTGCTCATT AGTTCTGTTG GTATTCTTGT CTGTTTGATC
1021 ACAACCCTAT TTGCTACGGA TTTGTTCGAA ATCAAGGTTG TCAAGGAAAT TGAACCAGCT
1081 TTGAAGAAGC AGCTCATCAT CTCCACTATT CTAATGACAG TAGGAATTGC AATTGTTACT
1141 TGGATTGGTG TGCCATCTTC CTTCACCATT TACAATTTTG GGGTTCAGAA AGTTGTTAAG
1201 AATTGGCAAC TATTTTTGTG CGTGGGTGTT GGTCTCTGGG CTGGACTTAT TATTGGTTTT
1261 GTTACTGAGT ACTACACCAG CAATGCTTAC AGCCCTGTGC AGGATGTTGC CGACTCTTGC
1321 AGGACGGGAG CAGCAACCAA TGTTATATTT GGCCTTGCTT TAGGATACAA ATCTGTGATC
1381 ATCCCAATTT TTGCCATAGC AATCAGTATT TTTGTTAGTT TTAGCTTTGC TGCTATGTAT
1441 GGGATTGCAG TTGCTGCCCT TGGAATGTTG AGCACCATTG CAACTGGGTT GGCTATCGAT
1501 GCTTATGGTC CCATCAGTGA CAATGCTGGA GGTATAGCTG AGATGGCTGG TATGAGCCAT
1561 CGCATTCGTG AGAGAACTGA TGCTCTTGAT GCTGCTGGAA ACACCACTGC TGCCATTGGA
1621 AAGGGATTTG CCATTGGGTC AGCGGCGTTG GTGTCTTTGG CTCTATTTGG TGCCTTTGTG
1681 AGCCGTGCTG CTATCACGAC CGTAGATGTC TTGACCCCAA AAGTTTTCAT TGGTTTAATA
1741 GTTGGAGCCA TGCTTCCTTA CTGGTTCTCT GCAATGACCA TGAAGAGTGT GGGAAGTGCT
1801 GCTTTAAAGA TGGTTGAGGA GGTTCGCAGA CAGTTCAACA CCATTCCCGG TCTCATGGAG
1861 GGTCATGCCA AGCCAGATTA TGCTACCTGT GTCAAAATCT CAACTGATGC CTCTATCAAG
1921 GAGATGATCC CTCCTGGTGC CCTTGTTATG CTTACTCCCC TCATTGTTGG AACTTTCTTT
1981 GGTGTAGAGA CTCTTTCTGG TGTCCTTGCT GGGGCTCTTG TTTCCGGTGT TCAGATTGCA
2041 ATTTCTGCAT CCAATACTGG TGGGGCATGG GACAATGCCA AGAAGTACAT TGAGGCCGGT
2101 GTGTCCGAGC ATGCAAGGAC CCTAGGTCCA AAGGGATCCG ATCCTCACAA GGCAGCTGTC
2161 ATCGGTGACA CTGTTGGAGA CCCTCTCAAG GATACCTCCG GTCCATCACT TAACATTCTT
2221 ATCAAGCTTA TGGCAGTCGA GTCACTTGTG TTTGCTCCCT TCTTTGCCAC ACACGGTGGT
2281 CTACTCTTCA AGATCTTTTA G 实施例 3 GhAVPl-2基因植物表达载体构建
植物表达载体 rd29A- GhAVPl-2-2300构建流程如图 1所示。
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 NPTII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 Tnos作为 GhAVPl-2基因的启动子和终止子。 具体步骤简述如下：
SEQ ID NO: 11和 SEQ ID NO: 12以植物表达载体 PBI 121 (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 μ 1 PCR反应体系： 10 μ 1 5 XPS Buffer, 3 μ 1 2· 5mM的 dNTP， 1. 0 μ 1 PBI121, 1. 0 μ 1 PrimeSTAR、 10 μ Μ 的引物 SEQ ID NO: 11和 SEQ ID NO: 12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; 94 °C 变性 30 s， 56°C退火 30 s， 72 °C 延伸 30 s， 33个循环； 72 °C 延 伸 10 min。 PCR产物通过 EcoRI、 Bglll (购自 New England Biolabs )酶切连接到 pCAMBIA2300 获得 pCAMBIA2300-l， 连接反应条件见 pr omega T4连接酶盒所示步骤。
SEQ ID NO: 11 ：
GCAC GAATTC ATACAAATGGACGAACGGAT SEQ ID NO: 12：
ATCC AGATCT AGATCCGGTGCAGATTATTTG SEQ ID No: 13和 SEQ ID No : 14以 PBI121为模板扩增 Tnos，采用 TaKaRa的 PrimeSTAR
HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5mM的 dNTP， 1.0 μΐ PBI121， 1.0 ^ PrimeSTAR 10 μΜ的引物 SEQ ID NO: 13和 SEQ ID NO: 14各 2.0μ1， 以及 31 μ1的双 蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s, 72 °C 延伸 30 s， 33个循环； 72°C 延伸 10 min。 PCR产物通过 Sacl、 EcoRI (购自 New England Biolabs )酶切 连接到 pCAMBIA2300-l获得 pCAMBIA2300-2, 连接反应条件是 promega T4连接酶试剂盒所 示步骤。
SEQ ID No: 13：
AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID No: 14：
T AGAATTC CCAGTGAATT CCCGATCTAG TA
SEQ ID NO: 15和 SEQ ID NO: 16以拟南芥 (哥伦比亚型，购自 www.arabidopsis.org)DNA为模 板 (参考 Zeng I, 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)扩增拟南芥 rd29 A启动子。采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5><PS Buffer, 3 μΐ 2.5mM的 dNTP， 1.0 μΐ拟南芥 DNA, 1.0 ^ PrimeSTAR 10 μΜ的引物 SEQ ID NO: 15和 SEQ ID NO: 16各 2.0μ1， 以及 31 μΐ的双 蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s, 72 °C 延伸 30 s， 33 个循环； 72°C 延伸 10 min。 PCR产物通过 HindIII、 Sail (购自 New England Biolabs ) 酶切连接 到 pCAMBIA2300-2获得 pCAMBIA2300-3， 连接反应条件是 promega T4连接酶试剂盒所示步 骤。
SEQ ID No: 15：
ACTAAGCTT CCTTCTTGACATCATTCAATTTTA SEQ ID No: 16：
TGAgt cga cTCCAAAGATT TTTTTCTTTC CAATAG
SEQ ID No: 17和 SEQ ID No: 18扩增 AVP1-2基因， （模板是实施例 2所获得含有 AVP 1-2 基因的 pGEM T-easy重组载体）， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ 1 PCR反 应体系： 10 μ 1 5XPS Buffer, 3 μ 1 2· 5mM的 dNTP， 1. 0 μ 1 AVP1- 2- pGEM， 1. 0 μ 1 PrimeSTAR、 10 μ M的引物 SEQ ID NO: 17和 SEQ ID NO: 18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; 94 °C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 2min， 33个循环； 72 °C 延伸 10 min。 PCR产物通过 Sall、 Sac I (购自 New England Biolabs ) 酶切连接到 PCAMBIA2300-3, 获得植物表达载体 rd29A-GhAVPl-2_2300， 连接反应条件是 pr omega T4连 接酶试剂盒所示步骤。
SEQ ID No: 17：
TGAGTCGACATGGGGGCGGCGATGTTGTCGGAG SEQ ID No: 18：
AAGGAGCTC CTAAAAGATC TTGAAGAGTA GACCACC
实施例 4 rd29A-GhAVPl-2-2300表达载体转化农杆菌
农杆菌 LBA4404 (购自 Biovector Science Lab, Inc) 感受态细胞制备： 提前 l_2d将农 杆菌 LBA4404在含 50 μ g/ml利福平和 50 μ g/ml链霉素的 LB固体培养基平板（LB液体培养 基中加入 1.5%的琼脂）上划单斑接种， 28°C培养 1至 2d。挑取单菌落接种于 5ml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_16h)至 0D600值 为 0. 4， 形成种子菌液。 取 5ml活化后的菌液 （ 1： 20的比例）接种于 100ml同样浓度抗生素 的 LB液体培养基中， 28°C摇动培养 2-2. 5h至 0D600=0. 8。 冰浴菌液 10min， 每隔 3min摇匀 一次， 令细菌均匀进入休眠状态。 于 4°C下 4000g离心 10min， 弃上清液； 加入一定量预冷 10%甘油重悬浮菌体， 4°C下 4000g离心 10min， 收集沉淀； 用 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 ΐ/管将其分装， 于 -70°C保存备用。
转化农杆菌： 在冰上融化上述农杆菌 LBA4404感受态细胞， 往 40 μ 1的感受态细胞中加 入 1 μ 1的实施例 3制备的表达载体构建 rd29A-GhAVPl-2-2300， 混匀后冰浴约 10 min。 将 上述混合物用枪转移到预冷的电击杯中， 轻敲使混合物到达底部， 注意不要有气泡。 将电击 杯（购自 bio-rad， 型号 Micropulser)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. lcm的电击杯， 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液体培养基中。 快速而轻柔的用枪将细胞 打匀。将上述悬浮液转入 1. 5ml的离心管， 28°C， 225rpm培养 lh。取 100-200 μ 1的菌液涂 布与相应的抗性筛选培养基 (LB固体培养基， 含 50μ§/ιη1利福平、 50μ§/ιη1链霉素、 50μ§/ιη1 卡那霉素）平板上， 28°C培养。 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（国家烟草中期库，获取单位中国农科院烟草所，库编号 I5A00660) 种子 30s， 再用 0.1%升汞浸泡 8min， 进行表面消毒。将消过毒的烟草种子置于 MS固体培养 ¾ (18.78 mMKN03, 1.25 mMKH2P04, 20.6 mMMliNOs, 1.5mMMgS04, 3.0mMCaCl2, 50μΜΚΙ, 100μΜΗ3ΒΟ3， 100 MMnSO4, 30 MZnSO4, 1 MNa2Mo04, 0.1 MCoCl2, 100 MNa2EDTA, 100 MFeSO4, 7.4 g/L琼脂， 蔗糖 30g/L) 上无菌发芽， 制备无菌苗。 取无菌苗叶片剪成 5mm X 5mm大小的叶盘， 用实施例 4中处于对数生长期的含表达载体的 农杆菌浸染叶盘 lOmin, 吸干菌液， 在黑暗条件下共培养 2天 （MS固体培养基)； 并以未被 农杆菌浸染的叶盘作为对照叶片。 将农杆菌浸染的叶片转到分化培养基 （MS 固体培养基 +lmg/L BA+0. lmg/L NAA+50mg/L卡那霉素 +500mg/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固体培养基 +50mg/L卡那霉素 +500mg/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 500mg/L头孢霉素的 MS固体培 养基上。 同时， 将对照叶片转到分化培养基 （MS 固体培养基 +lmg/L BA+O.lmg/L NAA+50mg/L卡那霉素 +500mg/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固体培养基 +50mg/L卡那霉素 +500mg/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 500mg/L头孢霉素的 MS固体培养基上。
取农杆菌浸染的烟草小苗叶片，提取基因组 DNA (同实施例 3中拟南芥 DNA提取方法) 后， 用引物 SEQIDNo: 17和 SEQIDNo: 18进行 PCR鉴定（Ex Taq购自 TAKARA, 50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ DNA, 1.0 μΐ Ex Taq、 10 μΜ 的引物 SEQIDNO: 17和 SEQIDNo: 18各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94 °C预变性 5min; 94 °C 变性 30 s， 58°C退火 30 s, 72 °C 延伸 2min， 33个循环； 72°C 延伸 lOmin), 选取 20株成功转化的转基因烟草编号成 T0M1-T0M20。
灭过菌的蛭石用 1/2MS培养基浸透，将上述培养 TQM1-TQM20转基因烟草小苗和对照小 苗移栽到所述蛭石上， 25°C、 10小时光培养 /14小时暗培养循环， 每 5天烧一次 1/2MS。 大 约 60天后获得种子。 实施例 6 过表达 GhAVPl-2转基因烟草 T1代的抗旱模拟实验及功能鉴定
灭过菌的蛭石用 1/2MS培养基浸透。将 TQ代转基因烟草 TQN1、 T0N2 T0N3 TQN4和 T0N5 的种子以及对照烟草种子分别播种在蛭石上， 25°C、 10小时光培养 /14小时暗培养循环， 每 5 天浇一次 1/2MS, 培养 25天之后， 摘取最下端一片叶子， 提取基因组 DNA (同实施例 3中拟南 芥 DNA提取方法）， 利用引物 SEQ ID NO: 17和 SEQ ID NO: 18做 PCR鉴定 (Ex Taq购自 TAKARA, 50 μ1 ΡΟ反应体系： 5 μΐ 10xEx Buffer， 3 μΐ 2.5mM的 dNTP， 2.0 μΐ DNA, Ι.Ο μΙ Εχ Taq、 10 μΜ的引物 SEQ ID NO: 17和 SEQ ID No: 18各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR反应条 件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s, 72 °C 延伸 2min， 33个循环； 72 °C 延 伸 10 min), 剔除阴性植株 （对照烟草也同样摘取一片叶子）。 挑取大小一致的转基因烟草 ( ΤιΝ ΤιΝ2 丁^3、 和 各 10株，编号分别是丁^^至！^^^- 、 TW2-1至 1^^2-10、 TW3-1至 1^^3-10、 TW4-1至 TiN^lO和 TW5-1至 1^^5-10)、 对照烟草 （10株， 编号是对照 1 至对照 10) 做耐旱实验。 转基因烟草、 对照烟草干旱 14天 (不浇水)， 25°C、 10小时光培养 /14 小时暗培养循环。 然后取出植株对植株进行称重。 上述 1代转基因植株的抗旱性鉴定表明， 对照植株都萎蔫严重，而转基因植株都能够正常生长，显示出明显的抗旱性（参见图 3及表 1 )。 在图 3中， 选取转基因植 ttTiNl-3和对照烟草 9示例性显示， 其他烟草的结果与它们类似。 实施例 7 过表达 GhAVPl-2转基因烟草 T1代的抗盐模拟实验及功能鉴定
灭过菌的蛭石用 1/2MS培养基浸透。 将 TQ代转基因烟草 TQN1、 T0N2 T0N3 TQN4和 T0N5 的种子以及对照烟草种子分别播种在蛭石上， 25°C、 10小时光培养 /14小时暗培养循环， 每 5天 浇一次 1/2MS, 培养 25天之后， 摘取最下端一片叶子， 提取基因组 DNA (同实施例 3中拟南芥 DNA提取方法），利用引物 SEQ ID NO: 17和 SEQ ID NO: 18做 PCR鉴定 (Ex Taq购自 TAKARA, 50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ DNA, 1.0 μ1 Εχ Τα 10 μΜ 的引物 SEQ ID NO: 17和 SEQ ID No: 18各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变 性 5 min; 94 °C 变性 30 s， 58°C退火 30 s, 72 °C 延伸 2min， 33个循环； 72°C 延伸 10 min)， 剔 除阴性植株 （对照烟草也同样摘取一片叶子）。 挑取大小一致的转基因烟草 （ TiNl、 T N2、 TiN3 和 各 10株， 编号分别是！^^^-^至！1^^。、 ΤιΝ2-1ΐ ΤιΝ2-20 TW3-11至 TiN3-20 TW4-11至 TW4-20和 TW5-11至 1^^5-20)、对照烟草（10株，编号是对照 11至对照 20) 做耐盐实验， 浇灌 lOO mMNaCl, 25°C、 10小时光培养 /14小时暗培养循环， 14天后观察结果： 代转基因植株的耐盐性鉴定表明， 对照植株都不能正常生长， 生长明显受到抑制， 而转基因 植株生长都明显高于对照植株， 显现出明显的耐盐性（参见图 4及表 1 )。 在图 4中， 选取转基因 植株 TW4-12和对照烟草 12示例性显示， 其他烟草的结果与它们类似。 转化事件 代植株重量 （g) (平均值士标准差） 耐盐处理 耐旱处理
TiNl 0.76±0· 07 0.70±0· 05
ΤιΝ2 0.65 + 0. 04 0.51 ±0· 06
ΤιΝ3 0.59±0· 06 0.49±0· 05
TiN4 0.67±0. 06 0.55 ±0· 06
TiN5 0.73 ±0· 06 0.62 ±0· 06 对照烟草植株 0.30±0· 04 0.31 ±0· 04

Claims

权 利 要 求 书
1.一种蛋白， 所述蛋白是棉花 AVP1类蛋白， 其序列为 SEQ ID No: 1。
2. 编码权利要求 1所述的蛋白的核苷酸序列。
3. 权利要求 2所述的核苷酸序列， 其特征在于具有 SEQ ID NO: 2所示的核苷酸序列。
4. 一种重组表达载体， 其含有权利要求 2或 3所述的核苷酸序列并且所述核苷酸序列 与所述表达载体的表达控制序列可操作地连接； 优选地， 所述载体为图 2 中所示的 rd29A-GhAVPl-2-2300载体。
5. 一种重组细胞， 其含有权利要求 2或 3所述的基因序列、 权利要求 4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。
6. 一种改善植物耐盐性和 /或抗旱性的方法， 包括： 将权利要求 2或 3所述的核苷酸 序列或者权利要求 4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。
7. 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 2 或 3所述的核苷酸序列或者权利要求 4所述的重组表达载体的植物或植物组织。
8. 权利要求 7所述的方法， 其中所述植物是烟草。
9. 权利要求 1的蛋白、 权利要求 2或 3所述的核苷酸序列、 权利要求 4所述的重组表 达载体或者权利要求 5所述的重组细胞用于改善植物耐盐性和抗旱性以及用于植物育种的 用途， 优选， 所述植物是烟草。
10. 权利要求 9所述的用途， 其中所述植物是烟草。
PCT/CN2012/076617 2012-06-08 2012-06-08 棉花avp1蛋白及其编码基因与应用 WO2013181832A1 (zh)

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