WO2014101153A1 - Cotton isopentenyl transferase ipt2, gene for encoding same, and application thereof - Google Patents

Cotton isopentenyl transferase ipt2, gene for encoding same, and application thereof Download PDF

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WO2014101153A1
WO2014101153A1 PCT/CN2012/087957 CN2012087957W WO2014101153A1 WO 2014101153 A1 WO2014101153 A1 WO 2014101153A1 CN 2012087957 W CN2012087957 W CN 2012087957W WO 2014101153 A1 WO2014101153 A1 WO 2014101153A1
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plant
seq
gene
expression vector
tobacco
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PCT/CN2012/087957
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Chinese (zh)
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王建胜
王君丹
田大翠
林余
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创世纪转基因技术有限公司
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Priority to CN201280078040.7A priority Critical patent/CN104884619B/en
Priority to PCT/CN2012/087957 priority patent/WO2014101153A1/en
Publication of WO2014101153A1 publication Critical patent/WO2014101153A1/en

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    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)

Definitions

  • the present invention relates to plant proteins and their coding genes and applications, and in particular to a cotton-derived isopentenyl transferase (IPT2) protein and a gene encoding the same, and the use thereof in breeding transgenic plants with improved drought resistance .
  • IPT2 isopentenyl transferase
  • the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid areas account for about 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.
  • a first aspect of the invention provides a gene encoding a prenyltransferase IPT2 of cotton (designated herein as Gh IPT2) having the sequence 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-Gh IPT2-2300 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 of GMPT2 (rd29A-GhIPT2-2300;
  • Figure 2 is a plasmid map of the plant expression vector Crd29A-GhIPT2-2300 of GMPT2.
  • Figure 3 shows the drought resistance of control tobacco and transgenic tobacco (Fig. 3a before drought, Fig. 3b after drought); CK (left): control tobacco; T1Q4 (right): transgenic tobacco lines.
  • FIG. 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
  • 9 is a plasmid PCR positive control
  • 10-13 is a drought-tolerant control tobacco plant.
  • 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:
  • a subtractive library was constructed by inhibition subtractive hybridization using the method shown by Clontech's PCR-selectTM cDNA Subtraction Kit.
  • the mRNA extracted from the leaves of the drought-treated cotton seedlings was used as a tester during the experiment, and the mRNA extracted from the leaves of the untreated cotton seedlings was used as a driver.
  • the specific steps are as follows:
  • 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.
  • the test seedlings were divided into 2 groups, 4 pots per group and 1 pot per pot.
  • the first group was a control group, which was cultured at 25 °C, photoperiod 16h light/8h dark (light intensity 2000-3000 Lx), and was normally watered.
  • 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. 3 leaves, rapidly frozen with liquid nitrogen, at -70 ° C Store in the refrigerator.
  • the cotton leaves of the control group and the drought treatment group were respectively 0.5 g, and the total RNA of cotton leaves was extracted with a plant 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 poly A+ 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
  • Nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-select TM
  • the cDNA Subtraction Kit kit comes with PCR amplification of the bacterial solution, and 452 positive clones were obtained, and all positive clones were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • sequence was SEQ ID No: 3.
  • Sequence analysis indicated that the protein encoded by the sequence belonged to the isopentenyl transferase.
  • the full-length coding gene corresponding to the sequence of SEQ ID No: 3 was named GhIPT2, and its corresponding protein was named IPT2.
  • GMPT2 GSP1 SEQ ID No: 4:
  • 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 the universal AUAP (provided with the kit), using mRNA reverse transcribed cDNA as a template. Specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 ⁇ 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: 4 And AUAP each 2.0 ⁇ 1, and 35 ⁇ 1 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, elongation at 72 °C for 1 min), extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50-fold with double distilled water, and 2.0 ⁇ l was used as a template, and the second round of PCR amplification was carried out by using SEQ ID NO: 5 and the universal primer AUAP.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 l OXEx Buffer 3 ⁇ 12.5 mM dNTP, 2.0 ⁇ 1 dilution
  • the first round of PCR product 1.0 ⁇ ⁇ Taq 10 ⁇ primers SEQ ID NO: 5 and AUAP each 2.0 ⁇ l, 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 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • a band of approximately 400 bp in the second PCR product (Gel Extraction Kit was purchased from OMEGA) was recovered and ligated into pGEM-T Easy Vector, and then transformed into Escherichia coli JM109 (specific method as above).
  • Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use.
  • SEQ ID NO: 5 and universal primer AUAP to carry out bacterial PCR amplification, 4 positive clones were obtained, and 4 positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the 3' end of the cDNA of the gene was obtained. .
  • GMPT2 GSP3 SEQ ID No: 6:
  • GMPT2 GSP4 SEQ ID No: 7:
  • GMPT2 GSP5 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).
  • the first round of PCR amplification was carried out using SEQ ID NO: 7 and the universal primer AAP (provided with the kit), and the cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 6) was used as a template for the first round of PCR amplification.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 ⁇ 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: 7 Double-distilled water of 2.0 ⁇ 1 and 35 ⁇ each with AAP.
  • 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 2.0 ⁇ l was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 8 and primer AUAP.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ l l OXEx Buffer 3 ⁇ 12.5 mM dNTP, 2.0 ⁇ l diluted first round PCR product, 1.0 ⁇ ⁇ Taq 10 ⁇ primer SEQ ID NO: 8 and AUAP each 2.0 ⁇ 1 , and 35 ⁇ 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.
  • a strip of approximately 800 bp fragment in the second PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated to pGEM-T Easy Vector, then converted to JM109 (the specific method is the same as above).
  • Ten white colonies were randomly picked and inoculated into LB liquid medium containing 50 ⁇ g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use.
  • the resulting 5 ' RACE product was cloned and sequenced, and spliced with the 3 ' RACE product sequencing result and the SEQ ID No: 3 sequence.
  • the GMPT2 full-length cDNA sequence was obtained SEQ ID No: 9:
  • the GMPT2 full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11.
  • the PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
  • 50 ⁇ 1 PCR reaction system 10 ⁇ 1 5 X PS Buffer 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ 1 cDNA, 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ M primer SEQ ID NO: 10 and P SEQ ID NO: 11 2.0 ⁇ 1, and
  • 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 PCR amplification product plus A tail 2.5 times the volume of absolute ethanol was added to the PCR product, placed at -20 ° C for 10 minutes, centrifuged, the supernatant was removed, dried, and then dissolved in 21 ⁇ ⁇ double distilled water. Then, 2.5 ⁇ ⁇ ⁇ ⁇ ⁇ Buffer 0.5 ⁇ 1 5 mM dATP, 1.0 ⁇ 1 Ex Taq was added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained DNA fragment of about 900 bp was recovered (Omega recovery kit), and ligated to the pGEM T-easy vector (GhIPT2-pGEM plasmid was obtained), and then transformed into JM109 (method as above).
  • 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 rd29A promoter and terminator Tnos were selected as promoters and terminators of the GMPT2 gene, respectively.
  • Primer SEQ ID NO: 12 and SEQ ID NO: 13 were used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase.
  • 50 yl PCR reaction system 10 l 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 ⁇ 121 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ ⁇ primer SEQ ID NO: 12 and P SEQ ID NO: 13 each 2.0 ⁇ 1, and 31 ⁇ ⁇ 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 ligated by EcoRI and Bglll (promega, T4 ligase cassette) to pCAMBIA2300 to obtain pCAMBIA2300-1.
  • Amplification of Tnos with pBI121 as a template using primers SEQ ID NO: 14 and SEQ ID NO: 15 using TaKaRa PrimeSTARHS DNA Polymerase 50 ⁇ PCR reaction system: 10 l5 XPSBuffer, 3 ⁇ 12.5 mM dNTP, 1.0 ⁇ 1 pBI121 1.0 ⁇ 1 Prime STAR, 10 ⁇ M primers SEQ ID NO: 14 and SEQ ID NO: 15 each 2.0 ⁇ 1 , and 31 ⁇ Double steamed 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 resulting PCR product was ligated by SacI, EcoRI digestion (promegaT4 ligase cassette) to pCAMBIA2300-1 to obtain pCAMBIA2300-2.
  • the Arabidopsis thaliana rd29A promoter was amplified using primers SEQ ID NO: 16 and SEQ ID NO: 17 with Arabidopsis thaliana (Columbia type, available from www.arabidopsis.org) as a template (see Zeng J., et al. 2002, Preparation of total DNA from "recalcitrant plant taxa", Acta Bot. Sin., 44(6): Method 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase from TaKaRa was used.
  • PCR reaction system 10 yl5 XPSBuffer, 3 ⁇ 12.5 mM dNTP, 1.0 ⁇ 1 Arabidopsis DNA, 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 ⁇ 1 , and 31 ⁇ 1 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 resulting PCR product was ligated by HindIII and Pstl (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3.
  • GMPT2 encoding gene was amplified with primers SEQ ID NO: 18 and SEQ ID NO: 19 (template was the positive GhIPT2-pGEM plasmid obtained in Example 2) using TaKaRa's PrimeSTARHS DNA polymerase.
  • 50 l PCR reaction system 10 l 5XPS Buffer, 3 ⁇ 12.5 mM dNTP, 1.0 ⁇ 1 GhIPT2-pGEM 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ ⁇ primer SEQ ID NO: 18 and P SEQ ID NO: 19 each 2.0 ⁇ 1, and 31 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (94 V denaturation 30 s, 58 ° C annealing 30 s, 72 V extension 2 min), 72 V extension 10 min.
  • the obtained PCR product was ligated by Pstl and Sad (the ligation method is the same as above) to pCAMBIA2300-3, and the plant expression was obtained. rd29A-GhIPT2-2300.
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 in LB solid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml streptomycin 1-2 days in advance Single spotted inoculation, cultured 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.
  • Centrifuge at 4000 g for 10 min at 4 ° C discard the supernatant; add a certain amount of ice-cold 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 ° C, collect the precipitate; pre-cool with ice 10 % glycerol was washed 3-4 times repeatedly; then the bacterial pellet was resuspended by adding 10% glycerol pre-cooled in an appropriate amount of ice bath, dispensed in 40 ⁇ M/tube, and stored at -70 °C until use.
  • Transformation of Agrobacterium The competent cells were thawed on ice, and 1 ⁇ l of plasmid was added to 40 ⁇ l of competent cells, mixed and ice bathed for about 10 min. A mixture of competent cells and rd29A-GhIPT2-2300 plasmid DNA was transferred to an ice-cold electric shock cup (purchased from bio-md) using a pipette, and tapped to bring the suspension to the bottom of the electric shock cup, taking care not to have air bubbles. Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup on the base electrode of the shock chamber.
  • an ice-cold electric shock cup purchased from bio-md
  • the program of MicroPulser (purchased from bio-rad) is set to "Agr" and the electric shock is once. Immediately remove the electric shock cup and add LB medium pre-warmed at 28 °C. Quickly and gently spread the cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C for 225 rpm for 1 h. 100-200 ⁇ l of the bacterial solution is 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 the sterile seedlings were cut into 5 mm X 5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing the expression vector rd29A-GhIPT2-2300 in the logarithmic growth phase for 10 min, and the bacteria were sucked in the dark condition.
  • Co-culture for 2 days (MS solid medium). Transfer the leaves to a differentiation solid medium (MS+1 mg/1 cytokinin (BA) + 0.1 mg/1 naphthaleneacetic acid (NAA) + 50 mg/1 kanamycin + 500 mg/1 cephalosporin) , incubated with 2000 Lx of light for 16 hours per day for about 45 days.
  • a differentiation solid medium MS+1 mg/1 cytokinin (BA) + 0.1 mg/1 naphthaleneacetic acid (NAA) + 50 mg/1 kanamycin + 500 mg/1 cephalosporin
  • the buds are transferred and transferred to rooting solid medium (MS+50 mg/1 kanamycin + 500 mg/1 cephalosporin) for cultivation. After about a day, after the root system was developed, the seedlings were transferred to MS solid medium supplemented with 500 mg/1 cephalosporin for number storage.
  • rooting solid medium MS+50 mg/1 kanamycin + 500 mg/1 cephalosporin
  • the leaves of the obtained transgenic tobacco plants were cut out, DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and PCR amplification was carried out using SEQ ID NO: 10 and SEQ ID NO: 11 (50 ⁇ l PCR reaction system). : 5 ⁇ 1 ⁇ ⁇ ⁇ BufFer 3 ⁇ 1 2.5 mM dNTP 2.0 ⁇ 1 DNA 1.0 ⁇ 1 Ex Taq, 10 ⁇ M primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 ⁇ 1 , and 35 ⁇ 1 Double-distilled water.
  • the sterilized vermiculite was soaked in 1/2 MS medium.
  • T0Q1—T0Q20 transgenic tobacco and control tobacco seeds were planted on vermiculite, 15 seeds per pot, 25 °C, 14 hours light culture/10 hours dark culture cycle.
  • 1/2MS was poured once a week, and after 25 days of culture, SEQ ID NO: 10 and SEQ ID NO: 11 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 of the same size were kept in each pot.
  • T1 transgenic plants plants grown from T0 transgenic plants
  • T1Q4, T1Q7, and T1Q9 lines showed significant drought resistance (see Figures 3a and 3b).
  • T1Q4 the results of T1Q7, T1Q9 are similar, not shown here).
  • the relative expression of IPT2 protein was detected by amplifying GhIPT2 by SEQ ID NO: 10 and P SEQ ID NO: 20.
  • the PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase.
  • 50 yl PCR reaction system 10 ⁇ 1 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ 1 cDNA, 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ M primer SEQ ID NO: 10 and P SEQ ID NO: 20 2.0 ⁇ 1, and 30 ⁇ ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 29 cycles (94 V denaturation 30 s, 58 °C annealing 30 s, 72 V extension lmin), 72 V extension 10 min.
  • M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.)
  • 1-8 is a drought-tolerant T1 transgenic tobacco plant
  • 9 is a plasmid PCR positive control (rd29A-GhIPT2).
  • 10-13 is a drought-tolerant control tobacco plant.
  • the size of the band shown is consistent with the size of GMPT2 (approximately 930 bp). The results showed that the transcription of GMPT2 was stronger in the drought-tolerant T1 transgenic tobacco plants, and there was no GMPT2 transcription in the drought-tolerant control tobacco plants.

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  • Plant Pathology (AREA)
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Abstract

Provided are an isopentenyl transferase (IPT2) from cotton, a gene for encoding same, and an application thereof in culturing transgenic plants with improved drought-tolerance.

Description

一种棉花异戊烯基转移酶 IPT2及其编码基因与应用  Cotton isopentenyl transferase IPT2 and its coding gene and application
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异戊烯 基转移酶 （IPT2) 蛋白及其编码基因， 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。 FIELD OF THE INVENTION The present invention relates to plant proteins and their coding genes and applications, and in particular to a cotton-derived isopentenyl transferase (IPT2) protein and a gene encoding the same, and the use thereof in breeding transgenic plants with improved drought resistance . BACKGROUND OF THE INVENTION Abiotic stresses, such as drought, salting, extreme temperatures, chemical pollution, and oxygen damage, can cause serious damage to the growth and development of plants and cause significant losses to crop yields. Among them, 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. According to statistics, the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid areas account for about 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.
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 transcriptional activators in abscisic acid signaling. Plant Cell, 15: 63-78. )。 Since the stress tolerance of plants is mostly quantitative, the available germplasm resources are scarce. It is very difficult to improve the stress tolerance of plants by conventional breeding techniques. It is especially difficult to cultivate true stress-tolerant varieties. In recent years, with the deepening of research on the molecular mechanism of plant stress resistance and the rapid development of molecular biology technology, stress resistance research has progressed from physiological level to molecular level, which promotes the development of plant stress resistance genetic engineering. When plants are stressed, they will respond accordingly 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 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. In recent years, studies on the ability of plants to improve stress tolerance through transgenic techniques, as well as studies on stress-tolerant crops, xerophytes and halophytes, have yielded significant results for stress-related genes and signal transduction. 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. 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.
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异戊烯基转移酶（本文命名为 IPT2)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。  However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress remain to be further studied. Research on the function and expression regulation of stress-responsive genes will provide an important basis for the study of plant stress-resistance related signaling pathways and signal transmission network systems. SUMMARY OF THE INVENTION The present inventors cloned a coding gene for an isopentenyltransferase (designated herein as IPT2) of cotton using SSH (Suppression Subtractive Hybridization) in combination with RACE (rapid amplification of cDNA ends) and determined Its DNA sequence. It has also been found that transgenic plants can significantly improve the drought resistance of transgenic plants after they are introduced into plants, and these traits can be stably inherited.
本发明第一方面提供棉花的一个异戊烯基转移酶 IPT2 的编码基因 （本文命名为 Gh IPT2), 其序列为 SEQ ID N0: 2。  A first aspect of the invention provides a gene encoding a prenyltransferase IPT2 of cotton (designated herein as Gh IPT2) having the sequence 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-Gh IPT2-2300载体。  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-Gh IPT2-2300 vector shown in Fig. 2.
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。  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.
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。  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; Preferably, the plant is tobacco.
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。  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 Preferably, the plant is tobacco.
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。  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; Preferably, the plant is tobacco.
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 GMPT2的植物表达载体 (rd29A-GhIPT2-2300;>的构建流程。 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. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a construction flow of a plant expression vector of GMPT2 (rd29A-GhIPT2-2300;
图 2是 GMPT2的植物表达载体 Crd29A-GhIPT2-2300)的质粒图。  Figure 2 is a plasmid map of the plant expression vector Crd29A-GhIPT2-2300 of GMPT2.
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； T1Q4 (右）： 转基因烟草株系。  Figure 3 shows the drought resistance of control tobacco and transgenic tobacco (Fig. 3a before drought, Fig. 3b after drought); CK (left): control tobacco; T1Q4 (right): transgenic tobacco lines.
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， 9为质粒 PCR 阳性对照， 10-13为不耐干旱对照烟草植株。 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：  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, 9 is a plasmid PCR positive control, and 10-13 is a drought-tolerant control tobacco plant. 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:
具体方法为：  The specific method is:
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：  A subtractive library was constructed by inhibition subtractive hybridization using the method shown by Clontech's PCR-selectTM cDNA Subtraction Kit. The mRNA extracted from the leaves of the drought-treated cotton seedlings was used as a tester during the experiment, and the mRNA extracted from the leaves of the untreated cotton seedlings was used as a driver. The specific steps are as follows:
( 1 ) 供试材料：  (1) Test materials:
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— 30cm时用于实验。 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 μ M KI, 100 μ MH 3 ) B0 3 , 100 μ M MnS0 4 , 30 μ M ZnS0 4 , 1 μ M Na 2 MoO 4 , 0.1 μM CoCl 2 , 100 μM Na 2 EDTA, 100 μ M FeS0 4 ) once. It was used for experiments when the seedlings were as high as 25-30 cm.
( 2 ) 材料处理：  (2) Material handling:
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。 The test seedlings were divided into 2 groups, 4 pots per group and 1 pot per pot. The first group was a control group, which was cultured at 25 °C, photoperiod 16h light/8h dark (light intensity 2000-3000 Lx), and was normally watered. 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. 3 leaves, rapidly frozen with liquid nitrogen, at -70 ° C Store in the refrigerator.
( 3 ) 总 RNA提取：  (3) Total RNA extraction:
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 poly A+ RNA from total RNA)分离 mRNA。 The cotton leaves of the control group and the drought treatment group were respectively 0.5 g, and the total RNA of cotton leaves was extracted with a plant 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 poly A+ RNA from total RNA.
( 4 ) 抑制差减杂交：  (4) Suppression of subtractive hybridization:
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。 Suppression Subtractive Hybridization performed by PCR-select TM cDNA Clontech's method shown Subtraction Kit kit. Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and 2 μg of Tester cDNA and 2 μg of Driver cDNA were used as starting materials for subtractive hybridization. The Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 h in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated. 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. .
( 5 ) cDNA差减文库的构建与初步筛选、 克隆、 鉴定  (5) Construction and preliminary screening, cloning and identification of cDNA subtraction library
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,另力 B 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。 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 μl PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 μ 1, 2 Χ Τ4 ligase buffer 5 μl, pGEM-T Easy vector 1 μl, T4 DNA ligase 1 μl, ligated overnight at 4 °C. Take 10 μΐ of the reaction product, add 100 μl of competent E. coli JM109C from TAKARA), ice bath for 30 min, heat shock for 60 seconds, ice bath for 2 min, and force B 250 μ 1 LB medium (containing 1% Tryptone (purchased from OXOID), 0.5% Yeast Extract (purchased from OXOID), 1% NaCl (purchased from Sinopharm)) was placed in a 37 ° C water bath, shaken at 225 rpm for 30 min, and inoculated with 200 μl of bacteria solution. The plates were incubated at 37 ° C for 18 h at LB (ibid) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA) containing 50 μg/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 μg/ml ampicillin, cultured overnight at 37 ° C, and glycerol was added to a final concentration of 20%, and stored at -80 ° C. spare. Nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-select TM The cDNA Subtraction Kit kit comes with PCR amplification of the bacterial solution, and 452 positive clones were obtained, and all positive clones were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing.
(6) 差异克隆的 cDNA测序分析：  (6) cDNA sequencing analysis of differential clones:
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 棉花异戊烯基转移酶基因 GMPT2的克隆  After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 405 effective ESTs were obtained (unigene example 2 clone of cotton isopentenyltransferase gene GMPT2)
克隆子 Gh-D2-141去掉冗余 DNA后， 序列为 SEQIDNo: 3， 序列分析表明该序 列编码的蛋白属于异戊烯基转移酶。 本文将 SEQ IDNo: 3序列对应的全长编码基因 命名为 GhIPT2， 其对应的蛋白命名为 IPT2。  After the cloned Gh-D2-141 was removed from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the protein encoded by the sequence belonged to the isopentenyl transferase. Here, the full-length coding gene corresponding to the sequence of SEQ ID No: 3 was named GhIPT2, and its corresponding protein was named IPT2.
SEQ ID No: 3：  SEQ ID No: 3:
1 A'CT C GASC CGAACCG TTS ACSAGA 3¾C.aACGTGC TAA¾C SC G CAAGAAGCAA 1 A'CT C GASC CGAACCG TTS ACSAGA 33⁄4C.aACGTGC TAA3⁄4C SC G CAAGAAGCAA
61 TCCAAGAaAT CAAAAGAAAC ACTTGCAGAT TA¾C TGTCG TCAATTGGAG AAGATTCAGC61 TCCAAGAaAT CAAAAGAAAC ACTTGCAGAT TA3⁄4C TGTCG TCAATTGGAG AAGATTCAGC
121 GGC GA'SGAA CAAGAASAAT GGACGA AC ACA.GGC GGA CGCCACCi'SAA GT C CA121 GGC GA'SGAA CAAGAASAAT GGACGA AC ACA.GGC GGA CGCCACCi'SAA GT C CA
131 GGCG GGAAA AGGAGCT'GAT GAS>5CTT&GG AAGAGC TGT AGCTGATCCA A¾~ 131 GGCG GGAAA AGGAGCT'GAT GAS>5CTT&GG AAGAGC TGT AGCTGATCCA A3⁄4~
GMPT2全长编码基因的克隆 Cloning of GMPT2 full-length coding gene
根据已经获得的 SEQIDNo: 3序列， 设计如下两条特异性引物， 作为 3' RACE 的 5' 端特异性引物。  Based on the sequence of SEQ ID No: 3 that has been obtained, the following two specific primers were designed as the 5'-end specific primer for 3' RACE.
GMPT2 GSP1: SEQ ID No: 4:  GMPT2 GSP1: SEQ ID No: 4:
TTAGACAAGA GACAACGTGC TA GMPT2 GSP 1： SEQ ID No : 5:  TTAGACAAGA GACAACGTGC TA GMPT2 GSP 1: SEQ ID No : 5:
TAAACTGCTG CAAGAAGCAA TC  TAAACTGCTG CAAGAAGCAA TC
实验步骤按试剂盒说明书操作 （ 3 ' RACE System for Rapid Amplification of cDNA Ends试剂盒购自 invitrogen公司）。  The experimental procedure was performed according to the kit instructions (3 'RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
用 SEQ ID NO: 4与通用弓 I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：  The first round of PCR amplification was carried out using SEQ ID NO: 4 and the universal AUAP (provided with the kit), using mRNA reverse transcribed cDNA as a template. Specific steps are as follows:
50 μ 1 PCR反应体系： 5 μ 1 ΙΟΧΕχ 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: 4和 AUAP各 2.0 μ1、 以及 35 μ 1 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。  50 μ 1 PCR reaction system: 5 μ 1 ΙΟΧΕχ Buffer 3 μ 12.5 mM dNTP, 2.0 μ 1 mRNA reverse transcribed cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primer SEQ ID NO: 4 And AUAP each 2.0 μ1, and 35 μ 1 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, elongation at 72 °C for 1 min), extension at 72 °C for 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: 5与 通用引物 AUAP进行第二轮 PCR扩增， 具体步骤如下：  The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μl was used as a template, and the second round of PCR amplification was carried out by using SEQ ID NO: 5 and the universal primer AUAP. The specific steps are as follows:
50 ylPCR反应体系： 5 μ 1 lOXEx Buffer 3 μ 12.5 mM的 dNTP、 2.0 μ 1稀释 的第一轮 PCR产物、 1.0 μ ΙΕχ Taq 10 μΜ的引物 SEQ ID NO: 5和 AUAP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 lmin)， 72 °C 延伸 10 min。 回收第二次 PCR产物中片 段约为 400 bp的条带（Gel Extraction Kit购自 OMEGA), 并将其连接于 pGEM-T Easy Vector, 然后转化到大肠杆菌 JM109(具体方法同上)。 随机挑取 10个白色菌落接种于 含有 50 g/ml 氨苄青霉素的 LB 液体培养基中， 37°C培养过夜后加甘油至终浓度 20%, -80°C保存备用。用 SEQIDNO: 5与通用引物 AUAP进行菌液 PCR扩增， 得到 4个阳性克隆， 将 4个阳性克隆送至英潍捷基 （上海） 贸易有限公司测序测序,获得该 基因的 cDNA的 3' 端。 50 ylPCR reaction system: 5 μ 1 l OXEx Buffer 3 μ 12.5 mM dNTP, 2.0 μ 1 dilution The first round of PCR product, 1.0 μ ΙΕχ Taq 10 μΜ primers SEQ ID NO: 5 and AUAP each 2.0 μl, and 35 μΐ 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. A band of approximately 400 bp in the second PCR product (Gel Extraction Kit was purchased from OMEGA) was recovered and ligated into pGEM-T Easy Vector, and then transformed into Escherichia coli JM109 (specific method as above). Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. Using SEQ ID NO: 5 and universal primer AUAP to carry out bacterial PCR amplification, 4 positive clones were obtained, and 4 positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the 3' end of the cDNA of the gene was obtained. .
根据已经获得的 GMPT2 基因片段， 设计如下三条特异性引物， 作为 5' RACE 的 3' 端特异性引物。  Based on the GMPT2 gene fragment that has been obtained, the following three specific primers were designed as the 3'-end specific primer for 5' RACE.
GMPT2 GSP3: SEQ ID No: 6:  GMPT2 GSP3: SEQ ID No: 6:
ACAAGCTCTT CCCAAGCCTC AT ACAAGCTCTT CCCAAGCCTC AT
GMPT2 GSP4: SEQ ID No: 7: GMPT2 GSP4: SEQ ID No: 7:
TGAGGAACAC TTCGGTGGCG TC TGAGGAACAC TTCGGTGGCG TC
GMPT2 GSP5: SEQ ID No: 8: GMPT2 GSP5: SEQ ID No: 8:
ATTCTTCTTG TTCCTCAGCC  ATTCTTCTTG TTCCTCAGCC
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）。  The experimental procedure was performed according to the kit instructions (5 'RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
用 SEQIDNO: 7与通用引物 AAP (试剂盒自带）， 以 mRNA逆转录的 cDNA (反 转录引物 SEQIDNO: 6) 为模板进行第一轮 PCR扩增， 具体步骤如下：  The first round of PCR amplification was carried out using SEQ ID NO: 7 and the universal primer AAP (provided with the kit), and the cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 6) was used as a template for the first round of PCR amplification. The specific steps are as follows:
50 μ 1 PCR反应体系： 5 μ 1 ΙΟΧΕχ 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: 7和 AAP各 2.0 μ1、 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， 55°C退火 30 s， 72 °C 延伸 lmin)， 72 °C 延伸 10 min。  50 μ 1 PCR reaction system: 5 μ 1 ΙΟΧΕχ Buffer 3 μ 12.5 mM dNTP, 2.0 μ 1 mRNA reverse transcribed cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primer SEQ ID NO: 7 Double-distilled water of 2.0 μ1 and 35 μΐ each with AAP. 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.
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: 8与 引物 AUAP进行第二轮 PCR扩增， 具体步骤如下：  The obtained PCR product was diluted 50 times with double distilled water, and 2.0 μl was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 8 and primer AUAP. The specific steps are as follows:
50 ylPCR反应体系： 5 μ 1 lOXEx Buffer 3 μ 12.5 mM的 dNTP、 2.0 μ 1稀释 的第一轮 PCR产物、 1.0 μ ΙΕχ Taq 10 μΜ的引物 SEQIDNO: 8和 AUAP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。 回收第二次 PCR产物中片 段约为 800bp的条带（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个白色菌落接种于含有 50 μ g/ml氨苄青霉素的 LB 液体培养基中， 37°C培养过夜后加甘油至终浓度 20%， -80 °C 保存备用。用 SEQ ID NO: 8与引物 AUAP进行菌液 PCR扩增（反应体系及反应条件 同上）， 得到 5个阳性克隆,选取其中 4个克隆送至英潍捷基 （上海） 贸易有限公司测 序测序,获得该基因的 cDNA的 5 ' 端。 50 ylPCR reaction system: 5 μl l OXEx Buffer 3 μ 12.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 μ ΙΕχ Taq 10 μΜ primer SEQ ID NO: 8 and AUAP each 2.0 μ 1 , and 35 μΐ 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. A strip of approximately 800 bp fragment in the second PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated to pGEM-T Easy Vector, then converted to JM109 (the specific method is the same as above). Ten white colonies were randomly picked and inoculated into LB liquid medium containing 50 μg/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. Using SEQ ID NO: 8 and primer AUAP for PCR amplification (reaction system and reaction conditions as above), 5 positive clones were obtained, and 4 clones were selected and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing. The 5' end of the cDNA of the gene was obtained.
所得的 5 ' RACE产物克隆测序后， 将其与 3 ' RACE产物测序结果以及 SEQ ID No: 3序列进行拼接。 获得 GMPT2全长 cDNA序列 SEQ ID No: 9：  The resulting 5 ' RACE product was cloned and sequenced, and spliced with the 3 ' RACE product sequencing result and the SEQ ID No: 3 sequence. The GMPT2 full-length cDNA sequence was obtained SEQ ID No: 9:
SEQ ID No: 9: SEQ ID No: 9:
1 CTTTGCTTGA TCATTCTTTG GACTAGAAAG GTTAATGATG ATGACAGTTT CAATGTCAAT 1 CTTTGCTTGA TCATTCTTTG GACTAGAAAG GTTAATGATG ATGACAGTTT CAATGTCAAT
61 GTGCAAAGAA ATGGTGTCTT TACTGGACAT AAGTTCTGGG GTGCTGAAAT CGGACTTCTT61 GTGCAAAGAA ATGGTGTCTT TACTGGACAT AAGTTCTGGG GTGCTGAAAT CGGACTTCTT
121 TAGTCCTAGG AGACATAAAG AAAAGGTTGT GATCCTAATG GGAGCAACCG GAACAGGAAA121 TAGTCCTAGG AGACATAAAG AAAAGGTTGT GATCCTAATG GGAGCAACCG GAACAGGAAA
181 ATCGAGGCTC TCCATTGATC TCGCAACCCG ATTCCCAGCA GAAATTATAA ATTCGGATAA181 ATCGAGGCTC TCCATTGATC TCGCAACCCG ATTCCCAGCA GAAATTATAA ATTCGGATAA
241 GA ACAAGTC CACGAAGGGC TTGACATAGT ACCAACAAG ATCAGTGAGG AAGAACGGTG241 GA ACAAGTC CACGAAGGGC TTGACATAGT ACCAACAAG ATCAGTGAGG AAGAACGGTG
301 TGGGATTCCG CACCATTTGT TAGGTGTAAT AAGTCCCAAC ATAGATTTTA CAGTGACGAA301 TGGGATTCCG CACCATTTGT TAGGTGTAAT AAGTCCCAAC ATAGATTTTA CAGTGACGAA
361 TTTCGTGGAC ATGGCTTCGC GTGCCACAGC TTCGATCGTG TCCCGCAGCC AGCTTCCGAT361 TTTCGTGGAC ATGGCTTCGC GTGCCACAGC TTCGATCGTG TCCCGCAGCC AGCTTCCGAT
421 CATAGCCGGC GGCTCGAATT CATACATTGA GGCTTTGGTC GATGACGAAA GGTCTAGATT421 CATAGCCGGC GGCTCGAATT CATACATTGA GGCTTTGGTC GATGACGAAA GGTCTAGATT
481 CCGGTCGAAA TATGAATGTT GTTTCCTTTG GGTAGATGTG GCAATGCCAG TGTTACATCA481 CCGGTCGAAA TATGAATGTT GTTTCCTTTG GGTAGATGTG GCAATGCCAG TGTTACATCA
541 GTATGTATCA GAGAGAGTAG ATAAGATGGT TGAAAATGGG ATGGTTGATG AGGCAAGAAG541 GTATGTATCA GAGAGAGTAG ATAAGATGGT TGAAAATGGG ATGGTTGATG AGGCAAGAAG
601 CTTCTTTGAT TACAATGCAA ATTATTCAAA GGGGATTAGG AAAGCTATCG GAGTCCCCGA601 CTTCTTTGAT TACAATGCAA ATTATTCAAA GGGGATTAGG AAAGCTATCG GAGTCCCCGA
661 ATTTGACCGG TACTTCCGAG CCGAACCGTT TTTAGACAAG AGACAACGTG CTAAACTGCT661 ATTTGACCGG TACTTCCGAG CCGAACCGTT TTTAGACAAG AGACAACGTG CTAAACTGCT
721 GCAAGAAGCA A CCAAGAAA TCAAAAGAAA CACTTGCAGA TTAGCTTGTC GTCAATTGGA721 GCAAGAAGCA A CCAAGAAA TCAAAAGAAA CACTTGCAGA TTAGCTTGTC GTCAATTGGA
781 GAAGATTCAG CGGCTGAGGA ACAAGAAGAA TTGGACGATA CACAGGCTCG ACGCCACCGA781 GAAGATTCAG CGGCTGAGGA ACAAGAAGAA TTGGACGATA CACAGGCTCG ACGCCACCGA
841 AGTGTTCCTC AGGCGTGGAA AAGGAGCTGA TGAGGCTTGG GAAGAGCTTG TAGCTGATCC841 AGTGTTCCTC AGGCGTGGAA AAGGAGCTGA TGAGGCTTGG GAAGAGCTTG TAGCTGATCC
901 AAGTACAGAA ATAGTAGCAC AATTTCTATG TGATATCAGC TCAGGAGCAC TGCTCAGTAC901 AAGTACAGAA ATAGTAGCAC AATTTCTATG TGATATCAGC TCAGGAGCAC TGCTCAGTAC
961 TAGTCCTGCC ATTGAGTTCC TTGTGGTATA ATGCTGTAAA ACCATATCTG ACCCAGGAAT961 TAGTCCTGCC ATTGAGTTCC TTGTGGTATA ATGCTGTAAA ACCATATCTG ACCCAGGAAT
1021 AACTAGTAAA GCCATAGATC TCAATGTTAA TTAACTTAAA ATAATGAAGA TATGCTACTT1021 AACTAGTAAA GCCATAGATC TCAATGTTAA TTAACTTAAA ATAATGAAGA TATGCTACTT
1081 AATGGAAGTC ACTTTTGGTG AAAAAGAAAT AGATACTTAG ATTTCATGGC CAAAAAAAAA1081 AATGGAAGTC ACTTTTGGTG AAAAAGAAAT AGATACTTAG ATTTCATGGC CAAAAAAAAA
1141 AAAAAAAAAA A 根据 SEQ ID NO:9序列设计一对引物如下： 1141 AAAAAAAAAA A A pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows:
SEQ ID No: 10:  SEQ ID No: 10:
ATGACAGTTT CAATGTCAAT GT SEQ ID No: 11 :  ATGACAGTTT CAATGTCAAT GT SEQ ID No: 11 :
TTATACCACA AGGAACTCAA TGG 通过 SEQ ID NO: 10和 SEQ ID NO: 11来克隆 GMPT2全长编码基因。 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 1 PCR反应体系： 10 μ 1 5 X PS Buffer 3 μ 1 2.5 mM的 dNTP、2.0 μ 1 cDNA、 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TTATACCACA AGGAACTCAA TGG The GMPT2 full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11. The PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR, 10 μM primer SEQ ID NO: 10 and P SEQ ID NO: 11 2.0 μ 1, and
30 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 lmin)， 72 °C 延伸 10 min。 30 μ ΐ 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.
PCR扩增产物加 A尾： PCR产物中加入 2.5倍体积的无水乙醇， -20°C放置 10分 钟， 离心， 去上清， 晾干， 然后用 21 μ ΐ双蒸水溶解。 然后向其中加入 2.5 μ ΐ ΙΟ Χ Εχ Buffer 0.5 μ 1 5 mM的 dATP、 1.0 μ 1 Ex Taq。 反应条件： 70°C反应 30分钟。 将得到 的约 900bp的 DNA片段回收（Omega回收试剂盒）， 并将其连接至 pGEM T-easy载体 上（得到 GhIPT2-pGEM质粒），然后转化 JM109(方法同上）。 随机挑取 10个白色菌落 接种于含有 50 g/ml氨苄青霉素的 LB 液体培养基中， 37°C培养过夜后加甘油至终浓 度 20%， -80°C保存备用。用 SEQ ID NO: 10与 SEQ ID NO: 11进行菌液 PCR扩增（反 应体系及反应条件同上）， 得到 7个阳性克隆,选取其中 4个阳性克隆送至英潍捷基（上 海）贸易有限公司测序,序列为 SEQ ID NO: 2，其编码的蛋白质的氨基酸序列为 SEQ ID NO: 1。  The PCR amplification product plus A tail: 2.5 times the volume of absolute ethanol was added to the PCR product, placed at -20 ° C for 10 minutes, centrifuged, the supernatant was removed, dried, and then dissolved in 21 μ ΐ double distilled water. Then, 2.5 μ ΐ ΙΟ Χ Εχ Buffer 0.5 μ 1 5 mM dATP, 1.0 μ 1 Ex Taq was added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained DNA fragment of about 900 bp was recovered (Omega recovery kit), and ligated to the pGEM T-easy vector (GhIPT2-pGEM plasmid was obtained), and then transformed into JM109 (method as above). Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. The bacterial liquid PCR amplification was carried out with SEQ ID NO: 10 and SEQ ID NO: 11 (the reaction system and the reaction conditions were the same as above), and 7 positive clones were obtained, and 4 positive clones were selected and sent to Yingji Jieji (Shanghai) Co., Ltd. The company was sequenced and the sequence is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is SEQ ID NO: 1.
IPT2蛋白的氨基酸序列： SEQ ID NO: 1 Amino acid sequence of IPT2 protein: SEQ ID NO: 1
1 MTVSMSMCKE MVSLLDISSG  1 MTVSMSMCKE MVSLLDISSG
21 VLKSDFFSPR RHKEKWILM  21 VLKSDFFSPR RHKEKWILM
41 GATGTGKSRL S IDLATRFPA  41 GATGTGKSRL S IDLATRFPA
61 EI INSDKIQV HEGLDIVTNK  61 EI INSDKIQV HEGLDIVTNK
81 ISEEERCGI P HHLLGVISPN  81 ISEEERCGI P HHLLGVISPN
101 IDFTVTNFVD MASRATAS IV  101 IDFTVTNFVD MASRATAS IV
121 SRSQLPI IAG GSNSYIEALV  121 SRSQLPI IAG GSNSYIEALV
141 DDERSRFRSK YECCFLWVDV  141 DDERSRFRSK YECCFLWVDV
161 AMPVLHQYVS ERVDK VENG  161 AMPVLHQYVS ERVDK VENG
181 MVDEARSFFD YNANYSKGIR  181 MVDEARSFFD YNANYSKGIR
201 KAIGVPEFDR YFRAEPFLDK  201 KAIGVPEFDR YFRAEPFLDK
221 RQRAKLLQEA IQEIKRNTCR  221 RQRAKLLQEA IQEIKRNTCR
241 LACRQLEKIQ RLRNKKNWTI  241 LACRQLEKIQ RLRNKKNWTI
261 HRLDATEVFL RRGKGADEAW  261 HRLDATEVFL RRGKGADEAW
281 EELVADPSTE IVAQFLCDIS  281 EELVADPSTE IVAQFLCDIS
301 SGALLSTSPA IEFLW* GMPT2编码基因的核苷酸序列 SEQ ID NO: 2 301 SGALLSTSPA IEFLW* Nucleotide sequence of GMPT2 encoding gene SEQ ID NO: 2
1 ATGACAGTTT CAATGTCAAT GTGCAAAGAA ATGGTGTCTT TACTGGACAT AAGTTCTGGG 1 ATGACAGTTT CAATGTCAAT GTGCAAAGAA ATGGTGTCTT TACTGGACAT AAGTTCTGGG
61 GTGCTGAAAT CGGACTTCTT TAGTCCTAGG AGACATAAAG AAAAGGTTGT GATCCTAATG61 GTGCTGAAAT CGGACTTCTT TAGTCCTAGG AGACATAAAG AAAAGGTTGT GATCCTAATG
121 GGAGCAACCG GAACAGGAAA ATCGAGGCTC TCCATTGATC TCGCAACCCG ATTCCCAGCA121 GGAGCAACCG GAACAGGAAA ATCGAGGCTC TCCATTGATC TCGCAACCCG ATTCCCAGCA
181 GAAATTATAA ATTCGGATAA GA ACAAGTC CACGAAGGGC TTGACATAGT ACCAACAAG181 GAAATTATAA ATTCGGATAA GA ACAAGTC CACGAAGGGC TTGACATAGT ACCAACAAG
241 ATCAGTGAGG AAGAACGGTG TGGGATTCCG CACCATTTGT TAGGTGTAAT AAGTCCCAAC241 ATCAGTGAGG AAGAACGGTG TGGGATTCCG CACCATTTGT TAGGTGTAAT AAGTCCCAAC
301 ATAGATTTTA CAGTGACGAA TTTCGTGGAC ATGGCTTCGC GTGCCACAGC TTCGATCGTG301 ATAGATTTTA CAGTGACGAA TTTCGTGGAC ATGGCTTCGC GTGCCACAGC TTCGATCGTG
361 TCCCGCAGCC AGCTTCCGAT CATAGCCGGC GGCTCGAATT CATACATTGA GGCTTTGGTC361 TCCCGCAGCC AGCTTCCGAT CATAGCCGGC GGCTCGAATT CATACATTGA GGCTTTGGTC
421 GATGACGAAA GGTCTAGATT CCGGTCGAAA TATGAATGTT GTTTCCTTTG GGTAGATGTG421 GATGACGAAA GGTCTAGATT CCGGTCGAAA TATGAATGTT GTTTCCTTTG GGTAGATGTG
481 GCAATGCCAG TGTTACATCA GTATGTATCA GAGAGAGTAG ATAAGATGGT TGAAAATGGG481 GCAATGCCAG TGTTACATCA GTATGTATCA GAGAGAGTAG ATAAGATGGT TGAAAATGGG
541 ATGGTTGATG AGGCAAGAAG CTTCTTTGAT TACAATGCAA ATTATTCAAA GGGGATTAGG541 ATGGTTGATG AGGCAAGAAG CTTCTTTGAT TACAATGCAA ATTATTCAAA GGGGATTAGG
601 AAAGCTATCG GAGTCCCCGA ATTTGACCGG TACTTCCGAG CCGAACCGTT TTTAGACAAG601 AAAGCTATCG GAGTCCCCGA ATTTGACCGG TACTTCCGAG CCGAACCGTT TTTAGACAAG
661 AGACAACGTG CTAAACTGCT GCAAGAAGCA A CCAAGAAA TCAAAAGAAA CACTTGCAGA661 AGACAACGTG CTAAACTGCT GCAAGAAGCA A CCAAGAAA TCAAAAGAAA CACTTGCAGA
721 TTAGCTTGTC GTCAATTGGA GAAGATTCAG CGGCTGAGGA ACAAGAAGAA TTGGACGATA721 TTAGCTTGTC GTCAATTGGA GAAGATTCAG CGGCTGAGGA ACAAGAAGAA TTGGACGATA
781 CACAGGCTCG ACGCCACCGA AGTGTTCCTC AGGCGTGGAA AAGGAGCTGA TGAGGCTTGG781 CACAGGCTCG ACGCCACCGA AGTGTTCCTC AGGCGTGGAA AAGGAGCTGA TGAGGCTTGG
841 GAAGAGCTTG TAGCTGATCC AAGTACAGAA ATAGTAGCAC AATTTCTATG TGATATCAGC841 GAAGAGCTTG TAGCTGATCC AAGTACAGAA ATAGTAGCAC AATTTCTATG TGATATCAGC
901 TCAGGAGCAC TGCTCAGTAC TAGTCCTGCC ATTGAGTTCC TTGTGGTATA A 实施例 3 GMPT2基因植物表达载体构建 901 TCAGGAGCAC TGCTCAGTAC TAGTCCTGCC ATTGAGTTCC TTGTGGTATA A Example 3 Construction of GMPT2 gene plant expression vector
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。 选择诱导型的 rd29A启动子及终止子 Tnos分别 作为 GMPT2基因的启动子和终止子。  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 ΝΡΤΠ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. . The inducible rd29A promoter and terminator Tnos were selected as promoters and terminators of the GMPT2 gene, respectively.
用引物 SEQ ID NO: 12和 SEQ ID NO: 13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 ρΒΙ121 1.0 μ 1 PrimeSTAR、 10 μ Μ的引物 SEQ ID NO: 12禾 P SEQ ID NO: 13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酶切将所得的 PCR产物连接 (promega, T4 连接酶盒）到 pCAMBIA2300获得 pCAMBIA2300-l。  Primer SEQ ID NO: 12 and SEQ ID NO: 13 were used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 yl PCR reaction system: 10 l 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 ρΒΙ121 1.0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO: 12 and P SEQ ID NO: 13 each 2.0 μ 1, and 31 μ ΐ 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 ligated by EcoRI and Bglll (promega, T4 ligase cassette) to pCAMBIA2300 to obtain pCAMBIA2300-1.
SEQ ID NO: 12  SEQ ID NO: 12
GCACGAATTC ggcgggaaac gacaatctga  GCACGAATTC ggcgggaaac gacaatctga
SEQ ID NO: 13  SEQ ID NO: 13
ATCCAGATCTAGATCCGGTGCAGATTATTTG  ATCCAGATCTAGATCCGGTGCAGATTATTTG
用引物 SEQ ID NO: 14和 SEQ ID NO: 15以 pBI121为模板扩增 Tnos，采用 TaKaRa 的 PrimeSTARHS DNA聚合酶。 50 μΐ PCR反应体系： 10 l5XPSBuffer、 3 μ 12.5 mM的 dNTP、 1.0 μ 1 pBI121 1.0 μ 1 Prime STAR、 10 μ M的引物 SEQ ID NO: 14和 SEQ ID NO: 15各 2.0 μ 1、 以及 31 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， 58°C退火 30 s， 72 °C 延伸 30 s)， 72 °C 延伸 10 min。 通 过 SacI、EcoRI酶切将所得的 PCR产物连接 (promegaT4 连接酶盒)到 pCAMBIA2300-l 获得 pCAMBIA2300-2。 Amplification of Tnos with pBI121 as a template using primers SEQ ID NO: 14 and SEQ ID NO: 15 using TaKaRa PrimeSTARHS DNA Polymerase. 50 μΐ PCR reaction system: 10 l5 XPSBuffer, 3 μ 12.5 mM dNTP, 1.0 μ 1 pBI121 1.0 μ 1 Prime STAR, 10 μM primers SEQ ID NO: 14 and SEQ ID NO: 15 each 2.0 μ 1 , and 31 μΐ Double steamed 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 resulting PCR product was ligated by SacI, EcoRI digestion (promegaT4 ligase cassette) to pCAMBIA2300-1 to obtain pCAMBIA2300-2.
SEQ ID NO: 14；  SEQ ID NO: 14;
AAGGAGCTCGAATTTCCCCGATCGTTCAA.A  AAGGAGCTCGAATTTCCCCGATCGTTCAA.A
SEQ ID NO: 1.5：  SEQ ID NO: 1.5:
TCAGAATTCCCAGTGAATICCCGATCTAGTA 用引物 SEQ ID NO: 16 和 SEQ ID NO: 17 以拟南芥 （哥伦比亚型 ， 购自 www.arabidopsis.org) DNA为模板扩增拟南芥 rd29A启动子（参考 Zeng J., et al.2002, Preparation of total DNA from " recalcitrant 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 yl5XPSBuffer、 3 μ 12.5 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: 16和 SEQ ID NO: 17各 2.0 μ 1、 以及 31 μ 1 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、 Pstl酶切将所得的 PCR产物连 接 （连接方法同上） 到 pCAMBIA2300-2获得 pCAMBIA2300-3。  TCAGAATTCCCAGTGAATICCCGATCTAGTA The Arabidopsis thaliana rd29A promoter was amplified using primers SEQ ID NO: 16 and SEQ ID NO: 17 with Arabidopsis thaliana (Columbia type, available from www.arabidopsis.org) as a template (see Zeng J., et al. 2002, Preparation of total DNA from "recalcitrant plant taxa", Acta Bot. Sin., 44(6): Method 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 μ 1 PCR reaction system: 10 yl5 XPSBuffer, 3 μ 12.5 mM dNTP, 1.0 μ 1 Arabidopsis DNA, 1.0 μ 1 PrimeSTAR, 10 μ Μ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μ 1 , and 31 μ 1 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 resulting PCR product was ligated by HindIII and Pstl (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3.
SEQ ID. NO; 16；  SEQ ID. NO; 16;
ACTAAGCTTGCTTCTIGACATCAITCAATTTTA SEQ ID; HO: 17- ACTAAGCTTGCTTCTIGACATCAITCAATTTTA SEQ ID; HO: 17-
TGACTGCAGTCCAAAGATTTTTTICTTTCCAATAG 用引物 SEQ ID NO: 18和 SEQ ID NO: 19扩增 GMPT2编码基因的全长序列 （模 板是实施例 2所获得阳性 GhIPT2-pGEM质粒）， 采用 TaKaRa的 PrimeSTARHS DNA 聚合酶。 50 lPCR反应体系： 10 l 5XPS Buffer、 3 μ 12.5 mM的 dNTP、 1.0 μ 1 GhIPT2-pGEM 1.0 μ 1 PrimeSTAR、 10 μ Μ的引物 SEQ ID NO: 18禾 P SEQ ID NO: 19 各 2.0 μ1、 以及 31 μΐ双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环 （94 V 变性 30 s， 58°C退火 30 s， 72V 延伸 2min)， 72V 延伸 10 min。 通过 Pstl、 Sad 酶切将所得的 PCR产物连接 （连接方法同上） 到 pCAMBIA2300-3， 获得植物表达载 体 rd29A- GhIPT2-2300。 TGACTGCAGTCCAAAGATTTTTTICTTTCCAATAG The full length sequence of the GMPT2 encoding gene was amplified with primers SEQ ID NO: 18 and SEQ ID NO: 19 (template was the positive GhIPT2-pGEM plasmid obtained in Example 2) using TaKaRa's PrimeSTARHS DNA polymerase. 50 l PCR reaction system: 10 l 5XPS Buffer, 3 μ 12.5 mM dNTP, 1.0 μ 1 GhIPT2-pGEM 1.0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO: 18 and P SEQ ID NO: 19 each 2.0 μ1, and 31 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 min, 33 cycles (94 V denaturation 30 s, 58 ° C annealing 30 s, 72 V extension 2 min), 72 V extension 10 min. The obtained PCR product was ligated by Pstl and Sad (the ligation method is the same as above) to pCAMBIA2300-3, and the plant expression was obtained. rd29A-GhIPT2-2300.
SEQ ID; NO : IS :  SEQ ID; NO : IS :
TGACTGCAG ATGACAGTTT CAATGTCAAT GT SEQ ID NO : 19；  TGACTGCAG ATGACAGTTT CAATGTCAAT GT SEQ ID NO : 19;
AAGGAGCTC TTATACCACA AGGAACTCAA TGG 实施例 4 rd29A-GhIPT2-2300表达载体转化农杆菌  AAGGAGCTC TTATACCACA AGGAACTCAA TGG Example 4 rd29A-GhIPT2-2300 Expression Vector Transformation Agrobacterium
农杆菌 LBA4404 (购自 Biovector Science Lab,Inc) 感受态细胞的制备： 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， 令所述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； 然后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。 Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 in LB solid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin 1-2 days in advance Single spotted inoculation, cultured at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 μg/ml rifampicin and 50 μg/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. 5 ml of activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin, and cultured at 28 ° C with shaking 2 2.5 h to OD 6QQ = 0.8. The ice bath liquid was shaken for 10 min every 3 min to allow the bacteria to enter the dormant state uniformly. Centrifuge at 4000 g for 10 min at 4 ° C, discard the supernatant; add a certain amount of ice-cold 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 ° C, collect the precipitate; pre-cool with ice 10 % glycerol was washed 3-4 times repeatedly; then the bacterial pellet was resuspended by adding 10% glycerol pre-cooled in an appropriate amount of ice bath, dispensed in 40 μM/tube, and stored at -70 °C until use.
转化农杆菌： 在冰上融化所述的感受态细胞，往 40 μ 1的感受态细胞中加入 1 μ 1 的质粒， 混匀后冰浴约 10 min。 将感受态细胞和 rd29A-GhIPT2-2300质粒 DNA的混 合物用移液枪转移到冰预冷的电击杯（购自 bio-md) 中， 轻敲使悬浮液到达电击杯底 部， 注意不要有气泡。 将所述电击杯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 而轻柔的用移液枪将细胞打匀。 将悬浮液转入 1.5 ml的离心管， 在 28°C 225 rpm摇 动培养 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  Transformation of Agrobacterium: The competent cells were thawed on ice, and 1 μl of plasmid was added to 40 μl of competent cells, mixed and ice bathed for about 10 min. A mixture of competent cells and rd29A-GhIPT2-2300 plasmid DNA was transferred to an ice-cold electric shock cup (purchased from bio-md) using a pipette, and tapped to bring the suspension to the bottom of the electric shock cup, taking care not to have air bubbles. Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup on the base electrode of the shock chamber. When using a 0.1cm size electric shock cup, the program of MicroPulser (purchased from bio-rad) is set to "Agr" and the electric shock is once. Immediately remove the electric shock cup and add LB medium pre-warmed at 28 °C. Quickly and gently spread the cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C for 225 rpm for 1 h. 100-200 μl of the bacterial solution is applied to the corresponding resistant selection medium plate (LB solid medium containing 50 μg/ml rifampicin, 50 μg/ml streptomycin, 50 μg/ 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
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固体培养基 （含 有 18.78 mM KN03 1.25 mM KH2P04、 20.6 mM H4N03 1.5 mM MgS04、 3.0 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、 7.4 g/1琼月旨， 30 g/1 蔗糖） 上于 无菌条件下发芽， 制备无菌苗。 取无菌苗叶片剪成 5 mm X 5 mm大小的叶盘， 用处于 对数生长期的含表达载体 rd29A-GhIPT2-2300的农杆菌浸染叶盘 10 min, 吸干菌液， 在黑暗条件下共培养 2天（MS固体培养基）。将叶片转到分化固体培养基（MS+1 mg/1 细胞分裂素 （BA) +0.1 mg/1萘乙酸 （NAA) +50 mg/1卡那霉素 +500 mg/1头孢霉素） 上， 每天用 2000 Lx的光照 16h， 培养 45天左右， 待芽长大后切下转移到生根固体培 养基（MS+50 mg/1卡那霉素 +500 mg/1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/1头孢霉素的 MS固体培养基上进行编号保存。 To soak tobacco seeds with 75% alcohol (National Tobacco Medium Term Bank, obtained by the Institute of Tobacco, Chinese Academy of Agricultural Sciences, library number I5A00660) 30 s, then wash twice with sterile double distilled water. Soak it in 0.1% liters of mercury for 8 min, then use Sterilize twice in steamed water to complete surface sterilization. Surface-sterilized tobacco seeds were placed in MS solid medium (containing 18.78 mM KN0 3 1.25 mM KH 2 P0 4 , 20.6 mM H 4 N0 3 1.5 mM MgS0 4 , 3.0 mM CaCl 2 , 50 μ M KI, 100 μ MH 3 B0 3 , 100 μ M MnS0 4 , 30 μ M ZnS0 4 , 1 μ M Na 2 Mo0 4 , 0.1 μ M CoCl 2 100 μ M Na 2 EDTA 100 μ M FeS0 4 , 7.4 g/1 Qiongyue, 30 g/1 sucrose) was germinated under sterile conditions to prepare sterile seedlings. The leaves of the sterile seedlings were cut into 5 mm X 5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing the expression vector rd29A-GhIPT2-2300 in the logarithmic growth phase for 10 min, and the bacteria were sucked in the dark condition. Co-culture for 2 days (MS solid medium). Transfer the leaves to a differentiation solid medium (MS+1 mg/1 cytokinin (BA) + 0.1 mg/1 naphthaleneacetic acid (NAA) + 50 mg/1 kanamycin + 500 mg/1 cephalosporin) , incubated with 2000 Lx of light for 16 hours per day for about 45 days. After the buds are grown, they are transferred and transferred to rooting solid medium (MS+50 mg/1 kanamycin + 500 mg/1 cephalosporin) for cultivation. After about a day, after the root system was developed, the seedlings were transferred to MS solid medium supplemented with 500 mg/1 cephalosporin for number storage.
剪取获得的转基因烟草植株的叶片， 提取 DNA (同实施例 3 中拟南芥 DNA提 取方法）， 用 SEQ ID NO: 10和 SEQ ID NO: 11进行 PCR扩增鉴定 （50 μ 1 PCR反应 体系： 5 μ 1 ΙΟ Χ Εχ BufFer 3 μ 1 2.5 mM的 dNTP 2.0 μ 1 DNA 1.0 μ 1 Ex Taq、 10 μ M 的引物 SEQ ID NO: 10和 SEQ ID NO: 11各 2.0 μ 1、 以及 35 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 2 min) , 72 °C 延伸 10 min)， 将 PCR鉴定为阳性植株编号为 T0Q1-T0Q20并保存。 实施例 6 过表达 GhIPT2转基因烟草 T1代植株的抗旱模拟实验  The leaves of the obtained transgenic tobacco plants were cut out, DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and PCR amplification was carried out using SEQ ID NO: 10 and SEQ ID NO: 11 (50 μl PCR reaction system). : 5 μ 1 ΙΟ Χ Εχ BufFer 3 μ 1 2.5 mM dNTP 2.0 μ 1 DNA 1.0 μ 1 Ex Taq, 10 μM primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 μ 1 , and 35 μ 1 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 2 min), extension at 72 °C for 10 min) PCR was identified as positive plant number T0Q1-T0Q20 and saved. Example 6 Overexpression of GhIPT2 Transgenic Tobacco T1 Generation Drought Resistance Simulation Experiment
灭过菌的蛭石用 1/2MS培养基浸透。 T0Q1— T0Q20转基因烟草及对照烟草的种 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: 10和 SEQ ID NO: 11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 T1Q4、 T1Q7、 T1Q9三个株系表现出明显的抗 旱性 （见图 3a和 3b， 以 T1Q4例， T1Q7、 T1Q9的结果与类似， 在此未示出）。 实施例 7 在转录水平上验证 IPT2蛋白表达  The sterilized vermiculite was soaked in 1/2 MS medium. T0Q1—T0Q20 transgenic tobacco and control tobacco seeds were planted on vermiculite, 15 seeds per pot, 25 °C, 14 hours light culture/10 hours dark culture cycle. 1/2MS was poured once a week, and after 25 days of culture, SEQ ID NO: 10 and SEQ ID NO: 11 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 of the same 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. The drought resistance of T1 transgenic plants (plants grown from T0 transgenic plants) showed that the control plants were wilting, while the T1Q4, T1Q7, and T1Q9 lines showed significant drought resistance (see Figures 3a and 3b). , in the case of T1Q4, the results of T1Q7, T1Q9 are similar, not shown here). Example 7 Verification of IPT2 protein expression at the transcriptional level
实施例 6中抗旱性好的 T1代转基因植株中随机选取 8棵 （分别属于上述三个抗 旱株系）， 实施例 6中对照植株随机选取 4棵， 各剪取干旱 14天的叶片 0.05 g， 用植 物 RNA提取试剂盒 （invitrogen) 提取总 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: 11 )。 通过 SEQ ID NO: 10禾 P SEQ ID NO: 20扩增 GhIPT2， 检测 IPT2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 μ 1 5 X PS Buffer, 3 μ 1 2.5 mM的 dNTP， 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR、 10 μ M的引物 SEQ ID NO: 10禾 P SEQ ID NO: 20 各 2.0 μ 1, 以及 30 μ ΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 29个循环 （94 V 变性 30 s, 58 °C退火 30 s, 72 V 延伸 lmin)， 72 V 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 PCR阳性对照 （rd29A-GhIPT2-2300质 粒）， 10-13为不耐干旱对照烟草植株。 图中所示条带大小与 GMPT2的大小一致 （约 为 930bp)。 结果表明，耐干旱 T1代转基因烟草植株中 GMPT2的转录较强， 不耐干旱 对照烟草植株中没有 GMPT2转录。 Among the transgenic plants of the T1 generation with good drought resistance in Example 6, 8 were randomly selected (respectively belong to the above three drought-tolerant strains), and the control plants in Example 6 were randomly selected from 4 plants, and the leaves of the drought-treated 14-day-old leaves were 0.05 g. Total RNA was extracted using a plant RNA extraction kit (invitrogen). UV spectrophotometric determination of total RNA at 260 nm The absorbance values at 280 nm were used to calculate individual RNA concentrations. Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription kit Superscript III Reverse Transcriptase (1 μg of total RNA as a template, reverse transcription primer SEQ ID NO: 11). The relative expression of IPT2 protein was detected by amplifying GhIPT2 by SEQ ID NO: 10 and P SEQ ID NO: 20. The PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase. 50 yl PCR reaction system: 10 μ 1 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR, 10 μM primer SEQ ID NO: 10 and P SEQ ID NO: 20 2.0 μ 1, and 30 μ ΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 29 cycles (94 V denaturation 30 s, 58 °C annealing 30 s, 72 V extension lmin), 72 V extension 10 min. The electrophoresis results of the product are shown in Figure 4: 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 PCR positive control (rd29A-GhIPT2). -2300 plasmid), 10-13 is a drought-tolerant control tobacco plant. The size of the band shown is consistent with the size of GMPT2 (approximately 930 bp). The results showed that the transcription of GMPT2 was stronger in the drought-tolerant T1 transgenic tobacco plants, and there was no GMPT2 transcription in the drought-tolerant control tobacco plants.
SEQ ID NO: 20:  SEQ ID NO: 20:
GGCAGGACTA GTACTGAGCA GT  GGCAGGACTA GTACTGAGCA GT

Claims

权 利 要 求 书 Claim
1. 棉花的一个异戊烯基转移酶的编码基因， 被命名为 GhIPT2， 其核苷酸序列如 SEQ ID NO: 2所示。 A gene encoding a prenyltransferase of cotton, designated as GhIPT2, having a nucleotide sequence as shown in SEQ ID NO: 2.
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。  2. A recombinant expression vector comprising the gene of claim 1 and the nucleotide sequence of the gene operably linked to an expression control sequence of the expression vector.
3. 权利要求 2所述的载体， 其为附图 2所示的 rd29A-GhIPT2-2300载体。  3. The vector of claim 2 which is the rd29A-GhIPT2-2300 vector shown in Figure 2.
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。  A recombinant cell comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3; preferably, the recombinant cell is a recombinant Agrobacterium cell.
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。  A method for improving drought resistance of a plant, comprising: introducing the gene of claim 1 or the recombinant expression vector of claim 2 or 3 into a plant or plant tissue and expressing the gene; preferably, the The plant is tobacco.
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。  A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3 under conditions effective to produce a plant.
7. 权利要求 6所述的方法， 其中所述植物是烟草。  7. The method of claim 6 wherein the plant is tobacco.
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。  The gene of claim 1, the recombinant expression vector of claim 2 or 3, or the recombinant cell of claim 4 for use in improving drought resistance of a plant and for use in plant breeding.
9. 权利要求 8所述的用途， 其中所述植物是烟草。  9. The use of claim 8 wherein the plant is tobacco.
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。  The gene-encoded protein according to claim 1, which has an amino acid sequence as shown in SEQ ID NO: 1.
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CN1408024A (en) * 1999-04-15 2003-04-02 卡尔根尼有限公司 Nucleic acid sequences to proteins involved in tocopherol synthesis
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CN1408024A (en) * 1999-04-15 2003-04-02 卡尔根尼有限公司 Nucleic acid sequences to proteins involved in tocopherol synthesis
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