WO2014101153A1 - Isopentényl transférase ipt2 de coton, gène codant pour celle-ci et application de celle-ci - Google Patents

Isopentényl transférase ipt2 de coton, gène codant pour celle-ci et application de celle-ci 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 PCT/CN2012/087957 priority Critical patent/WO2014101153A1/fr
Priority to CN201280078040.7A priority patent/CN104884619B/zh
Publication of WO2014101153A1 publication Critical patent/WO2014101153A1/fr

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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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  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une isopentényl transférase (IPT2) de coton, un gène codant pour celle-ci, et une application de celle-ci dans la culture de plantes transgéniques présentant une meilleure résistance à la sécheresse.
PCT/CN2012/087957 2012-12-31 2012-12-31 Isopentényl transférase ipt2 de coton, gène codant pour celle-ci et application de celle-ci WO2014101153A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2012/087957 WO2014101153A1 (fr) 2012-12-31 2012-12-31 Isopentényl transférase ipt2 de coton, gène codant pour celle-ci et application de celle-ci
CN201280078040.7A CN104884619B (zh) 2012-12-31 2012-12-31 一种棉花异戊烯基转移酶ipt2及其编码基因与应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2012/087957 WO2014101153A1 (fr) 2012-12-31 2012-12-31 Isopentényl transférase ipt2 de coton, gène codant pour celle-ci et application de celle-ci

Publications (1)

Publication Number Publication Date
WO2014101153A1 true WO2014101153A1 (fr) 2014-07-03

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PCT/CN2012/087957 WO2014101153A1 (fr) 2012-12-31 2012-12-31 Isopentényl transférase ipt2 de coton, gène codant pour celle-ci et application de celle-ci

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Country Link
CN (1) CN104884619B (fr)
WO (1) WO2014101153A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108753749B (zh) * 2018-06-20 2021-06-08 西南大学 家蚕tRNA异戊烯基转移酶基因及其重组载体和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1408024A (zh) * 1999-04-15 2003-04-02 卡尔根尼有限公司 编码参与生育酚合成的蛋白质的核酸序列
CN101061228A (zh) * 2004-09-17 2007-10-24 先锋高级育种国际公司 异戊烯基转移酶序列及其使用方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PL2324703T3 (pl) * 2005-03-21 2015-11-30 Univ California Sposoby poprawy wydajności zużycia wody w roślinie
EP2593554A2 (fr) * 2010-07-15 2013-05-22 Technion Research And Development Foundation Ltd. Construction d'acide nucléique destinée à accroître la tolérance au stress abiotique dans des végétaux

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1408024A (zh) * 1999-04-15 2003-04-02 卡尔根尼有限公司 编码参与生育酚合成的蛋白质的核酸序列
CN101061228A (zh) * 2004-09-17 2007-10-24 先锋高级育种国际公司 异戊烯基转移酶序列及其使用方法

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CN104884619A (zh) 2015-09-02
CN104884619B (zh) 2017-09-08

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