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WO2015042734A1 - Pyrophosphatase vacuolaire vp2 de bruguiera gymnorhiza, gène codant pour celle-ci, et application - Google Patents

Pyrophosphatase vacuolaire vp2 de bruguiera gymnorhiza, gène codant pour celle-ci, et application Download PDF

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Publication number
WO2015042734A1
WO2015042734A1 PCT/CN2013/001153 CN2013001153W WO2015042734A1 WO 2015042734 A1 WO2015042734 A1 WO 2015042734A1 CN 2013001153 W CN2013001153 W CN 2013001153W WO 2015042734 A1 WO2015042734 A1 WO 2015042734A1
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plant
seq
gene
expression vector
pcr
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Chinese (zh)
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崔洪志
何云蔚
王建胜
梁远金
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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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
    • C12N9/14—Hydrolases (3)

Definitions

  • the present invention relates to plant proteins and coding genes thereof and applications thereof, and in particular to a leaf vesicle pyrophosphatase VP2 derived from Phyllostachys pubescens and a coding gene thereof. And its use in the cultivation of transgenic plants with improved salt tolerance.
  • Salt stress is one of the most important abiotic stress hazards in agricultural production in the world. Salted soil is usually dominated by sodium salt, calcium salt or magnesium salt, and is a major factor affecting plant growth and causing food and economic crop yield reduction. The world's saline-alkali soil covers an area of about 400 million hectares, accounting for one-third of the irrigated farmland.
  • Saline-alkali land is widely distributed in China, and the existing saline-alkali land area is about 0.4 million hectares. With the increase of population in China and the reduction of cultivated land, the development and utilization of saline-alkali resources has extremely important practical significance.
  • the improvement of plant resistance to salt and alkali and the selection of plant species or strains suitable for growth on saline-alkali land with high economic and ecological value are economical and effective measures to utilize saline-alkali land.
  • most plants are poorly tolerant to saline and can only grow on soils with a sodium chloride content of less than 0.3%. Excess Na + in the soil will normalize the growth and metabolism of plants. Produces a toxic effect. Therefore, how to increase crop yield in a salted environment has become a very important issue in agricultural production worldwide.
  • the salt tolerance of plants is a very complex quantitative trait, and its salt tolerance mechanism involves various levels from plants to organs, tissues, physiology and biochemistry to molecules.
  • scientists from various countries have also done a lot of work for this purpose, and have made a lot of new progress, especially in the use of the model plant Arabidopsis to study the salt-tolerant molecular mechanism of plants, which has made a breakthrough in the research in this field ( Zhu JK. 2002. Salt and drought stress singal transduction in plants. Annu. Rev. Plant Biol. 53 : 1247-1273; Zhang ZL. 201 1.
  • a gene encoding a leaf follicle pyrophosphatase VP2 (designated herein as BgVP2) is provided, the sequence of which is SEQ ID NO: 2.
  • a second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, and the nucleotide sequence of the gene
  • the expression control sequence of the recombinant expression vector is operably linked; preferably, the expression vector is pCAMBIA2300 ; preferably, the recombinant expression vector is the 35S-BgVP2-2300 vector shown in Figure 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 invention provides a method for improving salt tolerance 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 Arabidopsis thaliana.
  • 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 Arabidopsis thaliana.
  • 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 salt tolerance of a plant and for use in plant breeding Use;
  • the plant is Arabidopsis thaliana.
  • the seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, the amino acid sequence of which is set forth in SEQ ID NO: 1.
  • FIG. la-lb The plant expression vector (35S-BgVP2-2300) of the map gene was constructed (Fig. la-lb).
  • Figure 23B is a plasmid map of the plant expression vector (35S-BgVP2-2300) of the Sg P2 gene.
  • Fig. 3 is a result of salt tolerance test of T1 generation Arabidopsis thaliana plants transgenic with B g VP2 gene, Tlp7 exhibits remarkable salt tolerance, and the results of ⁇ 1 ⁇ 8 and ⁇ 1 ⁇ 15 are similar thereto, and are not shown here.
  • Figure 4 shows the results of molecular level detection of the transcription level of the B g VP2 gene in T1 transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR.
  • M is DNA Ladder Marker (DL2000)
  • 1-8 is salt-tolerant T1 transgenic Arabidopsis plants (Tlp7, ⁇ 1 ⁇ 8, ⁇ 1 ⁇ 15, respectively)
  • 9-12 are non-transgenic control Arabidopsis plants.
  • Mulan was collected from Futian National Nature Reserve, Shenzhen, Guangdong province ( ⁇ 22° 53', E114° 01').
  • the P. guiem gymnoirhi hypocotyls with no pests, well-developed and mature levels were collected, and hypocotyls of similar size, length and weight were selected for the experiment.
  • plastic buckets (bottle diameter 18 cm, height 15 cm)
  • the bottom of each bucket is padded with plastic trays
  • the fine sand is river sand
  • the average particle size is about 1 mm
  • tap water is washed
  • each small barrel is planted with hypocotyls. 4.
  • Invitrogen extracts total RNA.
  • the absorbance of total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001.
  • the OD 260 / OD 280 ratio was 1.8-2.0, indicating that the total RNA purity was higher; 1.0% agarose was used to coagulate.
  • Gel electrophoresis detected the integrity of total RNA.
  • the 28S band was approximately twice as bright as the 18S band, indicating good RNA integrity.
  • mRNA was isolated using Qiagen's Oligotex mRNA Purification Kit (purified polyA+ RNA from total RNA).
  • the method according to Clontech's PCR-select TM cDNA Subtraction Kit kit instructions will be shown suppression subtractive hybridization.
  • Driver mRNA and Tester mRNA were reverse transcribed separately (reverse transcription primers were provided as primers) to obtain double-stranded cDNA, and 2 ⁇ g of Tester cDNA and 2 ⁇ g 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 hours 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 cDNA for the first forward subtractive hybridization.
  • the products of the two first forward subtractive hybridizations were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and the differentially expressed genes were amplified by two inhibitory PCR amplifications (PCR). Before, the second forward subtractive hybridization product is end-filled).
  • the second inhibitory PCR amplification product of the second forward subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) according to the instructions of the pGEM-T Easy kit (purchased from Promega)
  • the specific steps are linked to the pGEM-T Easy vector as follows: The following components are sequentially added to the 200 l PCR tube: Purified combined positive subtractive hybridization cDNA fragment second inhibitory PCR product 3 ⁇ 1 , 2 X T4 DNA ligase buffer 5 ⁇ l, pGEM-T Easy vector 1 ⁇ l, ⁇ 4 DNA ligase 1 ⁇ l, ligated overnight at 4 °C.
  • E. coli JM109 competent cells purchased from TAKARA
  • ice bath for 30 minutes heat shock for 60 seconds
  • ice bath for 2 minutes heat shock for 60 seconds
  • 250 l LB liquid medium was added.
  • tryptone purchased from OXOID
  • yeast extract purchased from OXOID
  • NaCl 1% NaCl
  • the nested PCR primers Primer 1 and Primer 2R (PCR-select TM cDNA Subtraction Kit from Clontech) were used to perform PCR amplification verification on the cultured cells, and 215 positive clones were obtained, and then all positive clones were sent.
  • Yingjie Jieji (Shanghai) Trading Co., Ltd. was sequenced.
  • sequence was SEQ ID No: 3.
  • Sequence analysis indicated that the protein encoded by the sequence belonged to leaf vesicular pyrophosphatase.
  • Sg P the full-length coding gene corresponding to the sequence of SEQ ID No: 3
  • VP2 the corresponding protein
  • BgVP2 GSP1 SEQ ID No: 4:
  • BgVP2 GSP2 SEQ ID No: 5:
  • the first round of PCR amplification was carried out using SEQ ID NO: 4 and the universal primer AUAP (provided with the kit), and the cDNA obtained by reverse transcription of the mRNA extracted by the salt treatment group was used as a template. Specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 ⁇ ⁇ ⁇ Buffer 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ 1 cDNA, 1.0 ⁇ 1 Ex Taq (purchased from TAKARA), 10 ⁇ M primer SEQ ID NO: 4 and AUAP each with 2.0 ⁇ 1 and 35 ⁇ 1 double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 2 minutes), 72 ° C for 10 minutes.
  • 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 lO X Ex Buffer 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ l diluted first round PCR product, 1.0 ⁇ 1 Ex Taq 10 ⁇ M primer SEQ ID NO: 5 and P AUAP Each of 2.0 ⁇ 1 and 35 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.
  • BgVP2 GSP3 SEQ ID No: 6:
  • BgVP2 GSP4 SEQ ID No: 7:
  • BgVP2 GSP5 SEQ ID No: 8:
  • the Ends kit was purchased from Invitrogen).
  • the cDNA obtained by reverse transcription of the mRNA extracted from the salt-treated group (reverse transcription primer SEQ ID NO: 6, dCTP plus tail) was used as a template.
  • PCR reaction system 5 ⁇ 1 ⁇ ⁇ ⁇ Buffer 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ 1 cDNA, 1.0 ⁇ 1 Ex Taq (purchased from TAKARA), 10 ⁇ M primer SEQ ID NO: 7 and P AAP each with 2.0 ⁇ l and 35 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.
  • the obtained PCR product was diluted 50 times with double distilled water, and 2.0 ⁇ ⁇ was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 8 and the primer AUAP.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 lO X Ex Buffer 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ l diluted first round PCR product, 1.0 ⁇ 1 Ex Taq 10 ⁇ M primer SEQ ID NO: 8 and P AUAP Each of 2.0 ⁇ 1 and 35 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (94 ° C denaturation
  • a pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows:
  • the Sg ⁇ full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11.
  • PCR was performed using TAKARA's PrimeSTAR HS DNA polymerase and cDNA obtained by reverse transcription of mRNA extracted from the salt-treated group.
  • 50 yl PCR reaction system 10 yl 5 X PS Buffer, 3 ⁇ l 2.5 mM dNTP, 2.0 ⁇ 1 cDNA, 1.0 ⁇ l PrimeSTAR HS DNA polymerase, 10 ⁇ M primers SEQ ID NO: 10 and SEQ ID NO: 11 each of 2.0 ⁇ 1 and 30 ⁇ 1 of double distilled water.
  • PCR reaction conditions Pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.
  • 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, air-dried, and then the resulting precipitate was dissolved in 21 ⁇ M of double distilled water. Then, 2.5 ⁇ l lO X Ex Buffer 0.5 ⁇ l 5 mM dATP, 1.0 l Ex Taq was added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained 2300 bp DNA fragment was recovered (Omega recovery kit), and ligated into pGEM T-easy vector to obtain BgVP2-pGEM plasmid, and then the ligation product was transformed into E.
  • coli JM109 competent cells (method same as above) The transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin, 40 g/mL X-gal, 24 g/mL IPTG for screening. 10 white colonies were randomly picked and inoculated in LB liquid medium containing 50 g/ml ampicillin. After incubation at 37 ° C overnight, glycerol was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C. .
  • Amino acid sequence of VP2 protein SEQ ID NO: 1
  • the plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ⁇ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. .
  • the 35S promoter and the Tnos terminator were selected as promoters and terminators of the BgVP2 gene, respectively.
  • the construction flow chart is shown in Figure 1.
  • Pnos was amplified using the plant expression vector pBI121 plasmid (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TAKARA's PrimeSTAR HS DNA polymerase.
  • 50 l PCR reaction system 10 l 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 ⁇ 121 plasmid, 1.0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ M primers SEQ ID NO: 12 and SEQ ID NO : 13 each of 2.0 ⁇ ⁇ and 31 ⁇ ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was digested with EcoRI, Bglll, and ligated into pCAMBIA2300 according to the kit instructions (Promega, T4 ligase kit) to obtain pCAMBIA2300-1.
  • Tnos was amplified using the primers SEQ ID NO: 14 and SEQ ID NO: 15 with the pBI121 plasmid as a template, using TAKARA's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was ligated by Kpnl, EcoRI digestion (Promega T4 ligase kit) to pCAMBIA2300-1 to obtain pCAMBIA2300-2.
  • TCAGAATTCCCAGTGAATTCCCGATCTAGTA The 35S promoter was amplified using the primers SEQ ID NO: 16 and SEQ ID NO: 17 using the pCAMBIA2300 plasmid as a template.
  • TAKARA's PrimeSTAR HS DNA polymerase was used. 50 ⁇ 1 PCR reaction system: 10 ⁇ 1 5 X PS Buffer 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ l pCAMBIA2300 plasmid, 1.0 ⁇ l PrimeSTAR HS DNA polymerase, 10 ⁇ M primer SEQ ID NO: 16 and P SEQ ID NO: 17 each of 2.0 ⁇ ⁇ and 31 ⁇ ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (94 ° C for 30 seconds, 58 ° C for 30 seconds, 72 ° C for 30 seconds), 72 ° C for 10 minutes.
  • the resulting PCR product was ligated by HindIII and Sail (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3.
  • TGAGTCGACAGAGATAGATTTGTAGAGAGACT The full-length sequence of the coding gene was amplified with primers SEQ ID NO: 18 and SEQ ID NO: 19 (the template was the positive BgVP2-pGEM plasmid obtained in Example 2) using TAKARA's PrimeSTAR HS DNA polymerase. 50 ⁇ ⁇ PCR reaction system: 10 l 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 BgVP2-pGEM plasmid, 1.0 ⁇ l PrimeSTAR HS DNA polymerase, 10 ⁇ M primer SEQ ID NO: 18 and SEQ ID NO: 19 each of 2.0 ⁇ ⁇ and 31 ⁇ ⁇ double distilled water.
  • PCR reaction conditions pre-variation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), and extension at 72 ° C for 10 minutes.
  • the obtained PCR product was ligated by Sall and Kpnl (connection method as above) to pCAMBIA2300-3, and the plant expression vector 35S-BgVP2-2300 was obtained after verification (Fig. 2).
  • Agrobacterium GV3101 (purchased from Shanghai Maiqi Biotechnology Co., Ltd.) Preparation of Competent Cells: Agrobacterium GV3101 was drawn on LB solid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin and 50 ⁇ ⁇ / ⁇ 1 gentamicin Single spot inoculation, culture at 28 °C for 1 to 2 days. Pick a single colony and inoculate 5 ml of LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin and 50 ⁇ ⁇ / ⁇ 1 gentamicin, and incubate overnight (about 12-16 hours) to OD 6 at 28 °C. . . A value of 0.4 forms a seed broth.
  • Transformation of Agrobacterium The GV3101 competent cells were thawed on ice, and 1 ⁇ M of the plasmid 35S-BgVP2-2300 obtained in Example 3 was added to 40 ⁇ M of the competent cells, and the mixture was mixed and ice-cooled for about 10 minutes. Transfer the mixture of competent cells after ice bath and 35S-BgVP2-2300 plasmid to a ice-cold 0.1 cm size electric shock cup (purchased from Bio-Rad) with a micropipette, tapping to bring the suspension to electric shock The bottom of the cup (be careful not to have bubbles). Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup to the base electrode of the electric shock chamber.
  • a ice-cold 0.1 cm size electric shock cup purchased from Bio-Rad
  • the GV3 101 Agrobacterium liquid of the transformed 35 S-Bg VP2-2300 expression vector obtained in Example 4 was inoculated to contain a 50 g/ml rifampicin, 50 ⁇ ⁇ / ⁇ 1 gentamicin, 50 g/ml card. Natamycin was cultured overnight in LB liquid medium, and inoculated 1:50 in the morning to contain rifampicin containing 50 g/ml, 50 ⁇ ⁇ / ⁇ 1 gentamicin, 50 g/ml kanamycin In the new LB medium (1 L), the culture was carried out for about 8 hours until the Agrobacterium liquid OD 6 (K) was between 1.0 and 1.2.
  • K Agrobacterium liquid OD 6
  • Seed disinfection Soak for 10 minutes with 70% ethanol, and occasionally suspend the seeds; then wash with sterile water four times, and occasionally suspend the seeds. Then, the treated seeds were uniformly coated on the surface of 1/2MS solid screening medium containing 50 ⁇ ⁇ / ⁇ 1 kanamycin (a maximum of 1500 seeds were seeded in a 150 mm diameter plate), and vernalized at 4 °C. After 2 days, it was cultured for 7-10 days at a constant temperature of 22 ° C, an illumination intensity of 3500-4000 k, and a photoperiod of 12 hours of darkness/12 hours of light. After germination of the transgenic seeds on the screening medium for 2 weeks, the plants capable of germination and normal growth were transferred to soil for further cultivation.
  • Each transformant of T0pl-T0pl9 and non-transgenic control Arabidopsis seeds were sown in 2 pots (20-30 seeds per pot). After sowing, the film is covered with a film to provide a moist environment for plant growth. Constant temperature 22 ° C, light intensity 3500-4000 lx, photoperiod of 12 hours dark / 12 hours light culture, 1/2 MS liquid medium per 7 days. After 25 days of culture, 1-2 leaves were cut per plant and DNA was extracted as a template, and PCR was carried out using SEQ ID NO: 18 and SEQ ID NO: 19 as primers (reaction system and conditions are the same as above). PCR-negative plants were removed, and 7-8 PCR-positive vaccines were retained in each pot.
  • Example 9 Salt-tolerant experiment of over-expressed transgenic Arabidopsis thaliana T 1 generation plants
  • Example 8 the transgenic Arabidopsis thaliana and the control Arabidopsis thaliana each retained a pot of plants for no treatment, normal watering
  • Example 9 Eight T1 transgenic plants with good salt tolerance in Example 9 were randomly selected (one of the above-mentioned Tlp7, Tlp8, and ⁇ 1 ⁇ 15 three salt-tolerant strains), and the control plants in Example 9 were randomly selected from 4 plants.
  • Salt 150 mM NaCl
  • Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase, and 1 ⁇ g of total RNA was used as a template for reverse transcription.
  • the B g VP2 fragment was amplified using primers SEQ ID NO: 10 and SEQ ID NO: 20 (SEQ ID NO: 20: CAAGAAGGAA ACAGTGCTGA AGAT), and its transcription was examined.
  • Primer SEQ ID NO: 21 SEQ ID NO: 21: 5-GCCATCCAAGCTGTTCTCTCTC
  • SEQ ID NO: 22 SEQ ID NO: 22: TTCTCGATGGAAGAGCTGGT
  • AtACT2 fragment Arabidopsis housekeeping gene: http: ⁇ www.ncbi.nlm.nih.gov/nuccore/AK317453.1
  • the PCR reaction was carried out using the Ex DNA polymerase of TAKARA and using the cDNA obtained by the above reverse transcription as a template.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 30 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.
  • the electrophoresis results of the PCR products are shown in Figure 4: 1-8 are salt-tolerant T1 transgenic Arabidopsis plants (three strains belonging to Tlp7, ⁇ 1 ⁇ 8, and ⁇ 1 ⁇ 15, respectively), and 9-12 are non-transgenic control Arabidopsis plants.
  • the results showed that B g VP2 was significantly transcribed in the salt-tolerant T1 transgenic Arabidopsis plants, and there was no transcription of BgVP2 in non-transgenic control Arabidopsis plants.

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Abstract

L'invention concerne la pyrophosphatase vacuolaire VP2 dérivée de Bruguiera gymnorhiza, un gène codant pour celle-ci, et une application correspondante, comprenant la culture d'une plante transgénique présentant une tolérance accrue au sel.
PCT/CN2013/001153 2013-09-25 2013-09-25 Pyrophosphatase vacuolaire vp2 de bruguiera gymnorhiza, gène codant pour celle-ci, et application Ceased WO2015042734A1 (fr)

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CN201380014011.9A CN105452452B (zh) 2013-09-25 2013-09-25 一种木榄液泡焦磷酸酶vp2及其编码基因与应用
PCT/CN2013/001153 WO2015042734A1 (fr) 2013-09-25 2013-09-25 Pyrophosphatase vacuolaire vp2 de bruguiera gymnorhiza, gène codant pour celle-ci, et application

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PCT/CN2013/001153 WO2015042734A1 (fr) 2013-09-25 2013-09-25 Pyrophosphatase vacuolaire vp2 de bruguiera gymnorhiza, gène codant pour celle-ci, et application

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CN106929492A (zh) * 2016-01-15 2017-07-07 中国林业科学研究院 唐古特白刺液泡膜H+‑PPase蛋白基因NtVP1、其编码蛋白、克隆方法

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