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WO2014205598A1 - Protéine de transport d'ions potassium haute affinité hkt1 obtenue à partir de thellungiella halophila, et gène codant et utilisation correspondante - Google Patents

Protéine de transport d'ions potassium haute affinité hkt1 obtenue à partir de thellungiella halophila, et gène codant et utilisation correspondante Download PDF

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WO2014205598A1
WO2014205598A1 PCT/CN2013/000751 CN2013000751W WO2014205598A1 WO 2014205598 A1 WO2014205598 A1 WO 2014205598A1 CN 2013000751 W CN2013000751 W CN 2013000751W WO 2014205598 A1 WO2014205598 A1 WO 2014205598A1
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plant
seq
gene
expression vector
pcr
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王建胜
田大翠
何云蔚
陈淼
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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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the present invention relates to plant proteins and their encoding genes and applications, and more particularly to a high affinity potassium ion transporter HKT1 derived from small salt mustard and a gene encoding the same, and in the cultivation of transgenic plants having improved salt tolerance application.
  • 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 billion 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 selection of plant resistance to salt and alkali, drought-tolerant ability and plant species or strains suitable for growth on saline-alkali land with high economic and ecological value is an economical and effective measure to utilize saline-alkali land. For most crops, most plants are poorly tolerant to saline and alkali, and can only grow on soil with a sodium chloride content of 0.3% or less. Excess Na + in the soil will be normal to the plant. Growth metabolism produces toxic effects. 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. 2011.
  • HKT1 high-affinity potassium ion transporter
  • SSH suppression subtractive hybridization
  • RACE rapid amplification of cDNA ends
  • the first aspect of the present invention provides a gene encoding a high affinity potassium ion transporter HKT1 of small salt mustard (herein named 73 ⁇ 4U7) having the sequence of 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-ThHKT1-2300 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.
  • Figure 1 shows the construction of the plant expression vector (35S-ThHKTl-2300) of the 73 ⁇ 4U7 gene (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector (35S-ThHKTl-2300) of the 73 ⁇ 4U7 gene.
  • Fig. 3 shows the salt tolerance test results of the T1 generation Arabidopsis thaliana plants transformed with the ThHKT1 gene, and Tle5 showed significant salt tolerance, and the results of TlelO and Tlel9 were similar thereto, which are not shown here.
  • Figure 4 shows the results of molecular level detection of the transcription level of ThHKT1 gene in 1 ⁇ generation 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 thaliana plants (Tle5, TlelO, Tlel9, respectively)
  • 9 is 35S-ThHKTl-2300 plasmid PCR positive control
  • 10- 13 is a non-transgenic control Arabidopsis plant.
  • Small salt mustard (TheUungieUa halophila, purchased from the Yanlan Plant Breeding Center of Ulan Buh and Desert Green Botanical Garden, Bayannao, Inner Mongolia, China) Seeded onto sterilized vermiculite, at 22 ° C, photoperiod 12 hours light / 12 hours dark (Light intensity 3000-4000 Lx) culture, 1/2MS liquid medium per week (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 ⁇ KI, 100 ⁇ H 3 B0 3 , 100 ⁇ MnS0 4 , 30 ⁇ ZnS0 4 , 1 ⁇ Na 2 Mo0 4 , 0.1 M CoCl 2 , 100 ⁇ Na 2 EDTA, 100 ⁇ FeS0 4 )-time. It was used for experiments when the seedlings reached a diameter of 5-6 cm.
  • the test plants were divided into 2 groups, 4 pots per group and 3 plants per pot.
  • the first group was the control group, which was normally watered with 1/2 MS liquid medium; the second group was the salt treatment group, and 1/2 MS liquid medium containing 300 mM NaCl was poured.
  • the culture was treated for 10 days, and then two groups of plants were collected in time (the roots were washed with steamed water), rapidly frozen with liquid nitrogen, and stored in a refrigerator at -70 °C.
  • the absorbance of total RNA at 260 nm and 280 nm, OD 26 was determined using a HITACHI UV spectrophotometer U-2001. /OD 28 .
  • the ratio of 1.8-2.0 indicates that the total RNA purity is high.
  • the integrity of total RNA is detected by 1.0% agarose gel electrophoresis.
  • the brightness of the 28S band is about twice that of the 18S band, indicating that the RNA integrity is good.
  • 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: The following components are sequentially added to the 200 ⁇ PCR tube: Purified combined positive subtractive hybridization cDNA fragment second inhibitory PCR product 3 ⁇ 1, 2xT4 DNA ligation Enzyme buffer 5 ⁇ 1, pGEM-T Easy vector 1 ⁇ 1, ⁇ 4 DNA ligase 1 ⁇ l, and 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 ⁇ liquid medium containing 1% tryptone (Tryptone, purchased from OXOID), 0.5% yeast extract (Yeast Extract, purchased from OXOID) and P 1% NaCl (purchased from Sinopharm) were placed in a shaker at 37 °C and oscillated at 225 rpm.
  • the white colonies picked were inoculated into LB liquid medium (same as above) containing 50 g/ml ampicillin in a 96-well cell culture plate (CORNING), and incubated at 37 ° C overnight to add glycerin to a final concentration of 20 glycerol. % (volume ratio), then stored at -80 ° C for later use.
  • the nested PCR primers Primer 1 and Primer 2R (PCR-selectTM cDNA Subtraction Kit from Clontech) were used to perform PCR amplification on the cultured cells, and 342 positive clones were obtained, and then all positive clones were sent.
  • Yingjie Jieji (Shanghai) Trading Co., Ltd. was sequenced.
  • ThHKTl GSP1 SEQ ID No: 4: ATTTACTCTC CGTTCGCCTT TG
  • ThHKTl GSP2 SEQ ID No : 5:
  • the experimental procedure was performed according to the kit instructions (3' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • the first round of PCR amplification was carried out using SEQ ID NO: 4 and the universal primer AUAP (provided with the kit), and the cDNA obtained by reverse transcription of the mRNA extracted from the salt-treated group was used as a template. Specific steps are as follows:
  • 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 1 minute), extension at 72 ° C for 10 minutes.
  • 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 by using SEQ ID NO: 5 and the universal primer AUAP.
  • the specific steps are as follows:
  • 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 1 minute), extension at 72 ° C for 10 minutes.
  • a strip of about 700 bp in the second round of PCR product was recovered (Gel Extraction Kit was purchased from OMEGA), and ligated into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (the method is the same as above)
  • the transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin and 40 ⁇ g mL X-gaK 24 g/mL IPTG for screening.
  • Ten white colonies were randomly picked and inoculated into LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 ampicillin, and cultured overnight at 37 ° C, glycerin was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C. spare.
  • ThHKTl GSP3 SEQ ID No : 6:
  • ThHKTl GSP4 SEQ ID No : 7: ACAGCTGTGA AGACCGAGAA GG
  • ThHKTl 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).
  • 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 55 ° 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 (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.
  • a band of about 600 bp in the second round of PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (the specific method is the same as above)
  • the transformed bacterial solution was applied to LB solid medium containing 50 ⁇ l of ampicillin and 40 ⁇ g of mL X-gaK 24 g/mL IPTG for screening.
  • Ten 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. .
  • ThHKT1 full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11.
  • the PCR reaction was carried out using the cDNA of the above-mentioned small salt mustard using the PrimeSTAR HS DNA polymerase of TAKARA as a template.
  • 50 ⁇ PCR reaction system 10 ⁇ 5xPS Buffer 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTARHS DNA polymerase, 10 ⁇ of primers SEQ ID NO: 10 and P SEQ ID NO:
  • 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 the resulting precipitate was dissolved in 21 ⁇ l of double distilled water. Then, 2.5 ⁇ ⁇ Buffer 0.5 ⁇ 5 mM dATP, 1.0 lExTaq was added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained 1600 bp DNA fragment was recovered (Omega recovery kit), and ligated into the pGEM T-easy vector to obtain a ThHKT1-pGEM plasmid, and then the ligated product was transformed into E.
  • coli JM109 competent cells (method as above) The transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin, 40 g/mL X-gal, and 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. .
  • 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 ThHKT1 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 ⁇ PCR reaction system: 10 ⁇ 5 xPS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ pBI121 plasmid, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 12 and SEQ ID NO: 13 each 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), and 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 using the pBI121 plasmid as a template, and TAKARA's PrimeSTAR HS DNA polymerase was used.
  • 50 ⁇ PCR reaction system 10 ⁇ 5 xPS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ pBI121 plasmid, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 14 and SEQ ID NO: 15 each 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 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 Sac I, EcoR I (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 with the pCAMBIA2300 plasmid as a template. TAKARA's PrimeSTAR HS DNA polymerase was used. 50 ⁇ PCR reaction system: 10 ⁇ 5 PS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ pCAMBIA 2300 plasmid, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 ⁇ and 31 ⁇ 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 30 seconds), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was ligated by Hind III, Pst I digestion (ligation method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3.
  • TGACTGCAGAGAGATAGATTTGTAGAGAGACT The full-length sequence of the 73 ⁇ 4U7-encoding gene was amplified with primers SEQ ID NO: 18 and SEQ ID NO: 19 (the template was the positive ThHKT1-pGEM plasmid obtained in Example 2) using TAKARA's PrimeSTAR HS DNA polymerase. 50 ⁇ PCR reaction system: 10 ⁇ 5 xPS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ ThHKTl-pGEM plasmid, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 18 and SEQ ID NO: 19 each 2.0 ⁇ and 31 ⁇ 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.
  • the obtained PCR product was ligated by Pstl and Sad (connection method as above) to pCAMBIA2300-3, and the plant expression vector 35S-ThHKTl-2300 was obtained after verification (Fig. 2).
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was spotted on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, 28 Incubate at °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin and 50 ⁇ ⁇ / ⁇ 1 streptomycin, and cultured overnight (about 12-16 hours) to OD 6 at 28 °C with shaking. . A value of 0.4 forms a seed broth.
  • Transformation of Agrobacterium The LBA4404 competent cells were thawed on ice, and 1 ⁇ M of the plasmid 35S-ThHKTl-2300 obtained in Example 3 was added to 40 ⁇ M of the competent cells, and the mixture was mixed and ice bathed for about 10 minutes. Transfer the mixture of competent cells after ice bath and 35S-ThHKTl-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 LBA4404 Agrobacterium liquid of the transformed 35S-ThHKTl-2300 expression vector obtained in Example 4 was inoculated to an LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 kanamycin (kan), and the next morning, 1 was pressed. :50 The cells were inoculated to a new LB medium (1 L) containing 50 ⁇ ⁇ / ⁇ 1 kanamycin and cultured for about 8 hours to Agrobacterium OD 6 . . Between 1.0 and 1.2.
  • 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.
  • Example 9 Salt tolerance test of transgenic Arabidopsis thaliana T1 plants overexpressing ThHKT1
  • transgenic Arabidopsis thaliana and control Arabidopsis thaliana each retained a pot of plants without treatment, and normal watering of 1/2 MS liquid medium, while simultaneously One pot of each plant was irrigated with 1/2 MS liquid medium containing 150 mM NaCl at a constant temperature of 22 ° C, light intensity of 3500-4000 k, 12 hours light culture / 12 hours dark culture cycle, and the results were observed after 14 days.
  • the salt tolerance of T1 transgenic plants plants grown from seeds of TO transgenic plants
  • the results of Tlel0 and Tlel9 are similar, not shown here).
  • Example 10 Verification of the expression of ThHKT1 gene at the transcriptional level
  • T1 transgenic plants resistant to salt in Example 9 were randomly selected (three salt-tolerant strains belonging to the above Tle5, TlelO and Tlel9, respectively), and the control plants in Example 9 were randomly selected from 4 plants. (150 mM NaCl) 0.05 g of leaves were treated for 14 days, and total RNA was extracted using a plant RNA extraction kit (Invitrogen). The absorbance values of total RNA obtained at 260 nm and 280 nm were determined by ultraviolet spectrophotometry, and the respective RNA concentrations were calculated.
  • Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay Ubox Superscript III Reverse Transcriptase, and 1 total RNA was used as a template, and the reverse transcription primer was SEQ ID NO: 11.
  • the ThHKT1 fragment was amplified using primers SEQ ID NO: 10 and SEQ ID NO: 20, and its transcription was examined.
  • PCR was carried out using TAKARA's PrimeSTAR HS DNA polymerase using the cDNA obtained by the above reverse transcription as a template.
  • 50 ⁇ l ⁇ Reaction system 10 ⁇ 5 > ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primer SEQ ID NO: 10 and P SEQ ID NO: 20 2.0 ⁇ l, and 30 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 32 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.
  • M is the DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), and 1-8 is the salt-tolerant T1 transgenic Arabidopsis plants (Tle5, Tlel0, Tlel9, respectively). Three strains), 9 is the 35S-ThHKTl-2300 plasmid PCR positive control, and 10-13 is the non-transgenic control Arabidopsis plant. The size of the band shown is the same as the size of the positive control (approximately 700 bp). The results showed that 73 ⁇ 4U7 of the salt-tolerant T1 transgenic Arabidopsis plants were significantly transcribed, and there was no transcription of ThHKT1 in non-transgenic control Arabidopsis plants.
  • SEQ ID NO: 20 GCCAGAGAAG ACCCGAGTTC TT

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Abstract

L'invention concerne une protéine de transport d'ions potassium haute affinité obtenue à partir de thellungiella halophila, un gène codant de celle-ci, et une utilisation de celle-ci pour cultiver une plante transgénique ayant une tolérance au sel améliorée.
PCT/CN2013/000751 2013-06-24 2013-06-24 Protéine de transport d'ions potassium haute affinité hkt1 obtenue à partir de thellungiella halophila, et gène codant et utilisation correspondante Ceased WO2014205598A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201380074518.3A CN105008388A (zh) 2013-06-24 2013-06-24 一种小盐芥高亲和钾离子转运蛋白hkt1及其编码基因与应用
PCT/CN2013/000751 WO2014205598A1 (fr) 2013-06-24 2013-06-24 Protéine de transport d'ions potassium haute affinité hkt1 obtenue à partir de thellungiella halophila, et gène codant et utilisation correspondante

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PCT/CN2013/000751 WO2014205598A1 (fr) 2013-06-24 2013-06-24 Protéine de transport d'ions potassium haute affinité hkt1 obtenue à partir de thellungiella halophila, et gène codant et utilisation correspondante

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