[go: up one dir, main page]

WO2014205597A1 - Hkt2 transporteuse présentant une affinité élevée pour les ions potassium et qui est dérivée de coton, gène codant et utilisation correspondante - Google Patents

Hkt2 transporteuse présentant une affinité élevée pour les ions potassium et qui est dérivée de coton, gène codant et utilisation correspondante Download PDF

Info

Publication number
WO2014205597A1
WO2014205597A1 PCT/CN2013/000750 CN2013000750W WO2014205597A1 WO 2014205597 A1 WO2014205597 A1 WO 2014205597A1 CN 2013000750 W CN2013000750 W CN 2013000750W WO 2014205597 A1 WO2014205597 A1 WO 2014205597A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
seq
gene
expression vector
plants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2013/000750
Other languages
English (en)
Chinese (zh)
Inventor
崔洪志
王建胜
王君丹
刘捷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BIOCENTURY TRANSGENE (CHINA) Co Ltd
Original Assignee
BIOCENTURY TRANSGENE (CHINA) Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BIOCENTURY TRANSGENE (CHINA) Co Ltd filed Critical BIOCENTURY TRANSGENE (CHINA) Co Ltd
Priority to CN201380074523.4A priority Critical patent/CN105189535A/zh
Priority to PCT/CN2013/000750 priority patent/WO2014205597A1/fr
Publication of WO2014205597A1 publication Critical patent/WO2014205597A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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 cotton-derived high affinity potassium ion transporter HKT2 and its encoding gene, 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 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 soils 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. 201 1.
  • HKT2 high affinity potassium ion transporter
  • the first aspect of the present invention provides a coding gene for a high affinity potassium ion transporter HKT2 of cotton (designated herein as GhHKT2) 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-GhHKT2-2300 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 is a construction flow of the plant expression vector (35S-GhHKT2-2300) of the G 3 ⁇ 4r2 gene (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector (35S-GhHKT2-2300) of the G 3 ⁇ 4r2 gene.
  • Fig. 3 is a salt tolerance test result of T1 generation Arabidopsis plants transgenic with GhHKT2 gene, Tlg7 exhibits remarkable salt tolerance, and the results of Tlgll and Tlgl3 are similar thereto, and are not shown here.
  • Figure 4 shows the results of molecular level detection of the transcription level of GhHKT2 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 plants (Tlg7, Tlgll, Tlgl3, respectively)
  • 9 is 35S-GhHKT2-2300 plasmid PCR positive control
  • 10- 13 is a non-transgenic control Arabidopsis plant.
  • African cotton (National Cotton Medium Term Bank, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) Seeded on sterilized vermiculite, cultured at 25 ° C, light dark cycle 16 h / 8 h, poured 1 per week /2MS liquid medium (containing 9.39 mM KN0 3 , 0.625 mM KH2P0 4 , 10.3 mM NH 4 N0 3 , 0.75 mMMgS0 4 , 1.5 mM CaCl 2 , 50 ⁇ , 100 ⁇ 3 ⁇ 3 , 100 MMnSO 4 , 30 MZnSO 4 , 1 ⁇ Na 2 Mo0 4 , 0.1 MCoCl 2 , 100 ⁇ Na 2 EDTA, 100 MFeSO 4 ) — times. It was used for experiments when the seedlings were as long as 25-30 cm.
  • test seedlings were divided into 2 groups of 4 plants each.
  • the first group was a control group, cultured at 25 ° C under light, placed in 1/2 MS liquid medium; the second group was treated with 25 ° C, light culture, and placed at a final concentration of 200 mM. Treatment in 1/2 MS liquid medium of NaCl for 6 hours. Cut the two groups in time after processing The roots of the seedlings were quickly frozen with liquid nitrogen and stored in a -70 ° C refrigerator.
  • 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.
  • Nested PCR primer Primer 1 and P Primer 2R were used to perform PCR amplification on the cultured cells, and 231 positive clones were obtained, and then all positive clones were obtained.
  • GhHKT2 GSP 1 SEQ ID No : 4:
  • GhHKT2 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 from the salt-treated 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 2.0 ⁇ ⁇ and 35 ⁇ ⁇ 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 1 minute), 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 58 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.
  • GhHKT2 GSP3 SEQ ID No : 6:
  • GhHKT2 GSP4 SEQ ID No : 7:
  • GhHKT2 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 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.
  • 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 primers SEQ ID NO: 7 and P AAP 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 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 ⁇ M was used as a template, and the second round of PCR amplification was carried out by using SEQ ID NO: 8 and the primer AUAP.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 l OXEx Buffer 3 ⁇ 12.5 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 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 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-TEasy 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.
  • a pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows:
  • the GhHKT2 full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11.
  • PCR was carried out using TAKARA's PrimeSTAR HS DNA polymerase using the cDNA of the above cotton as a template.
  • 50 l PCR reaction system 10 l 5XPS Buffer, 3 ⁇ 12.5 mM dNTP, 2.0 ⁇ 1 cDNA 1.0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ M primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 ⁇ 1 and 30 ⁇ 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 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 ⁇ l of OXEx Buffer 0.5 ⁇ 15 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 GhHKT2-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 and 40 g/mL X-gaK 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. .
  • SEQ ID NO: 10 and SEQ ID NO: 11 for bacterial liquid PCR amplification (reaction system and reaction conditions as above), 7 positive clones were obtained, and 4 positive clones were selected and sent to Yingjiejie (Shanghai) Trade. Sequencing, the resulting sequence is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is 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 GhHKT2 gene, respectively.
  • the construction flow chart is shown in Figure 1.
  • the plant expression vector pBI121 plasmid (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) was used as a template to amplify Pnos 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 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 yl 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 G 3 ⁇ 4 r2 encoding gene was amplified with primers SEQ ID NO: 18 and SEQ ID NO: 19 (template was the positive GhHKT2-pGEM plasmid obtained in Example 2), using TAKARA's PrimeSTAR HS DNA polymerase.
  • PCR reaction system 10 l 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 GhHKT2-pGEM plasmid, 1.0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ ⁇ primer SEQ ID NO: 18 and SEQ ID NO: 19 each of 2.0 ⁇ l and 31 ⁇ 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 2 minutes), 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-GhHKT2-2300 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-GhHKT2-2300 obtained in Example 3 was added to 40 ⁇ of the competent cells, and the mixture was mixed and ice-cooled for about 10 minutes. The mixture of the competent cells after the ice bath and the 35S-GhHKT2-2300 plasmid was transferred to an ice-cold 0.1 cm size electric shock cup (purchased from Bio-Rad) using a micropipette, and tapped to bring the suspension to an electric shock. The bottom of the cup (be careful not to have bubbles).
  • Example 5 Receptor Material Arabidopsis Culture Select the vermiculite with good water absorption and soft soil to match the nutrient soil (1:1) as the soil for Arabidopsis planting.
  • the LBA4404 Agrobacterium liquid of the transformed 35 S-GhHKT2-2300 expression vector obtained in Example 4 was inoculated to an LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 kanamycin (kan), and the next morning was pressed. 1 : 50 was inoculated into 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.
  • Each transformant of T0gl-T0g25 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 culturing for 25 days, 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 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 Tlg7, Tlgl and Tlgl3 salt-tolerant strains), and the control plants in Example 9 were randomly selected from 4 plants. Total leaves were treated with salt (150 mM NaCl) for 15 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 L1 box Superscript III Reverse Transcriptase, and 1 total RNA was used as a template, and the reverse transcription primer was SEQ ID NO: 11.
  • the GhffK fragment was amplified using primers SEQ ID NO: 10 and SEQ ID NO: 20 (SEQ ID NO: 20: ACTTCCAAAC CATGTTGTAT C), and its transcription was examined.
  • the PCR reaction was carried out using the TAKARA PrimeSTAR HS DNA polymerase using the cDNA obtained by the above reverse transcription as a template.
  • 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 2 minutes), 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 (Tlg7, Tlgl, respectively).
  • Tlg7, Tlgl, respectively Three strains of Tlgl3), 9 is a 35S-GhHKT2-2300 plasmid PCR positive control, and 10-13 are non-transgenic control Arabidopsis plants.
  • the size of the band shown is the same as the size of the positive control (approximately 1500 bp).
  • the results showed that G 3 ⁇ 4J2 was significantly transcribed in the salt-tolerant T1 transgenic Arabidopsis plants, and there was no transcription of GhHKT2 in non-transgenic control Arabidopsis plants.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne une HKT2 transporteuse présentant une affinité élevée pour les ions potassium et qui est dérivée de coton et le gène codant correspondant, ainsi que l'utilisation correspondante dans la culture d'une plante génétiquement modifiée présentant une tolérance améliorée au sel.
PCT/CN2013/000750 2013-06-24 2013-06-24 Hkt2 transporteuse présentant une affinité élevée pour les ions potassium et qui est dérivée de coton, gène codant et utilisation correspondante Ceased WO2014205597A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201380074523.4A CN105189535A (zh) 2013-06-24 2013-06-24 一种棉花高亲和钾离子转运蛋白hkt2及其编码基因与应用
PCT/CN2013/000750 WO2014205597A1 (fr) 2013-06-24 2013-06-24 Hkt2 transporteuse présentant une affinité élevée pour les ions potassium et qui est dérivée de coton, gène codant et utilisation correspondante

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2013/000750 WO2014205597A1 (fr) 2013-06-24 2013-06-24 Hkt2 transporteuse présentant une affinité élevée pour les ions potassium et qui est dérivée de coton, gène codant et utilisation correspondante

Publications (1)

Publication Number Publication Date
WO2014205597A1 true WO2014205597A1 (fr) 2014-12-31

Family

ID=52140733

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/000750 Ceased WO2014205597A1 (fr) 2013-06-24 2013-06-24 Hkt2 transporteuse présentant une affinité élevée pour les ions potassium et qui est dérivée de coton, gène codant et utilisation correspondante

Country Status (2)

Country Link
CN (1) CN105189535A (fr)
WO (1) WO2014205597A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113512563A (zh) * 2021-08-02 2021-10-19 中国农业科学院棉花研究所 GhPOT8基因在调控植物耐盐胁迫能力中的应用及调控植物耐盐胁迫能力的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110982815B (zh) * 2019-06-24 2023-03-31 河南科技学院 一种棉花钾转运体基因启动子及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006045829A1 (fr) * 2004-10-29 2006-05-04 Cropdesign N.V. Plantes à caractéristiques de croissance améliorées et procédé de production desdites plantes
WO2008129060A2 (fr) * 2007-04-23 2008-10-30 Basf Se Augmentation de la productivité d'une plante par combinaison d'agents chimiques avec des modifications transgéniques
CN101343316A (zh) * 2008-09-05 2009-01-14 中国科学院微生物研究所 一种钾离子转运相关蛋白系统及其编码基因簇与应用
CN101386854A (zh) * 2008-10-10 2009-03-18 哈尔滨师范大学 碱菀液泡膜Na+/H+反向转运蛋白的基因和氨基酸序列及基因制备方法和扩增引物
CN101456909A (zh) * 2009-01-13 2009-06-17 南京农业大学 一种大豆hkt类蛋白及其编码基因与应用
WO2012028646A1 (fr) * 2010-08-31 2012-03-08 Westfälische Wilhelms-Universität Münster Moyens destinés à améliorer des caractères agrobiologiques dans des plantes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102807990B (zh) * 2012-08-15 2014-05-14 中国农业科学院生物技术研究所 耐辐射异常球菌R1 trkA基因培育抗旱植物的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006045829A1 (fr) * 2004-10-29 2006-05-04 Cropdesign N.V. Plantes à caractéristiques de croissance améliorées et procédé de production desdites plantes
WO2008129060A2 (fr) * 2007-04-23 2008-10-30 Basf Se Augmentation de la productivité d'une plante par combinaison d'agents chimiques avec des modifications transgéniques
CN101343316A (zh) * 2008-09-05 2009-01-14 中国科学院微生物研究所 一种钾离子转运相关蛋白系统及其编码基因簇与应用
CN101386854A (zh) * 2008-10-10 2009-03-18 哈尔滨师范大学 碱菀液泡膜Na+/H+反向转运蛋白的基因和氨基酸序列及基因制备方法和扩增引物
CN101456909A (zh) * 2009-01-13 2009-06-17 南京农业大学 一种大豆hkt类蛋白及其编码基因与应用
WO2012028646A1 (fr) * 2010-08-31 2012-03-08 Westfälische Wilhelms-Universität Münster Moyens destinés à améliorer des caractères agrobiologiques dans des plantes

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113512563A (zh) * 2021-08-02 2021-10-19 中国农业科学院棉花研究所 GhPOT8基因在调控植物耐盐胁迫能力中的应用及调控植物耐盐胁迫能力的方法

Also Published As

Publication number Publication date
CN105189535A (zh) 2015-12-23

Similar Documents

Publication Publication Date Title
US20050193443A1 (en) Transcription factors, DNA and methods for introduction of value-added seed traits and stress tolerance
WO2013185258A1 (fr) Protéine hkt du coton et gène codant pour celle-ci, et leur utilisation
CN105073773B (zh) 一种小盐芥液泡膜焦磷酸酶vp1及其编码基因与应用
WO2015024143A1 (fr) Protéine à doigts de zinc zat10-1 provenant du coton, gène codant et applications associés
WO2015024142A1 (fr) Protéine à doigts de zinc azf2-1 provenant du coton, gène codant et applications associés
WO2014205597A1 (fr) Hkt2 transporteuse présentant une affinité élevée pour les ions potassium et qui est dérivée de coton, gène codant et utilisation correspondante
WO2015042734A1 (fr) Pyrophosphatase vacuolaire vp2 de bruguiera gymnorhiza, gène codant pour celle-ci, et application
WO2014205595A1 (fr) Atp hydrolase atpase-1 dérivée du coton, gène codant et utilisation de ceux-ci
WO2015024147A1 (fr) Protéine à doigts de zinc zpt5-5 du coton et gène codant et utilisations correspondantes
WO2014205598A1 (fr) Protéine de transport d'ions potassium haute affinité hkt1 obtenue à partir de thellungiella halophila, et gène codant et utilisation correspondante
WO2014205599A1 (fr) Antiport sodium-hydrogène de tonoplaste dans nhx3 dethellungiella halophila, gène codant et son utilisation
WO2014172825A1 (fr) Anti-protéine nhx2 sodium-hydrogène tonoplastique de thellungiella halophila, son gène codant et son utilisation
WO2015042740A1 (fr) Protéine de type calcineurine b cbl-4 de thellungiella halophila, gène codant pour celle-ci et application associée
WO2014063270A1 (fr) Protéine de la classe du canal ionique du coton, gène codant et leur utilisation
WO2014063271A1 (fr) Protéine de canal ionique du coton, gène codant et leurs utilisations
WO2015024145A1 (fr) Protéine à doigts de zinc zpt5-3 provenant du coton et son gène codant et ses utilisations
WO2015058323A1 (fr) Bétaïne aldéhyde déshydrogénase (badh) de bruguiear gymnorrhiza, gène codant pour celle-ci, et application du gène codeur
WO2015058322A1 (fr) Cofacteur à molybdène sulfurylase mcsu de bruguiera gymnorrhiza, gène codant pour celle-ci et utilisation
WO2014205596A1 (fr) Hydrolase atpase 2 atp du coton, gène codant et leur utilisation
WO2015058321A1 (fr) Monooxygénase cmo d'avicennia marina, gène codant et leur utilisation
WO2014172824A1 (fr) Protéine antiport sodium-hydrogène nhx1 tonoplaste issue de thellungiella halophila, gène codant pour celle-ci et son application
WO2015042732A1 (fr) Protéine de transport de sodium-hydrogène nah2 dérivée de kandelia candel, gène codant pour cette protéine, et applications correspondantes
WO2015042731A1 (fr) Protéine de transport de sodium-hydrogène nha1 dérivée de kandelia candel, gène codant pour cette protéine et application de cette protéine
WO2015042733A1 (fr) Protéine kinase cipk1 dérivée de bruguiera gymnorhiza, gène codant pour celle-ci, et son utilisation
WO2015042736A1 (fr) Pyrophosphatase vacuolaire vp1 issue de bruguiera gymnorhiza, gène codant celle-ci et utilisation du gène

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201380074523.4

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13887771

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13887771

Country of ref document: EP

Kind code of ref document: A1