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WO2014063271A1 - Protéine de canal ionique du coton, gène codant et leurs utilisations - Google Patents

Protéine de canal ionique du coton, gène codant et leurs utilisations Download PDF

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Publication number
WO2014063271A1
WO2014063271A1 PCT/CN2012/001426 CN2012001426W WO2014063271A1 WO 2014063271 A1 WO2014063271 A1 WO 2014063271A1 CN 2012001426 W CN2012001426 W CN 2012001426W WO 2014063271 A1 WO2014063271 A1 WO 2014063271A1
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plant
seq
gene
expression vector
cdna
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Chinese (zh)
Inventor
何云蔚
王建胜
王君丹
梁远金
梁丽
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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Priority to PCT/CN2012/001426 priority patent/WO2014063271A1/fr
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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 ion channel proteins and their coding genes and applications, and particularly to an ion channel protein SOS1 derived from cotton and a coding gene thereof, and the same Application in 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, drought-tolerant ability and the selection of 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.
  • most plants are poorly tolerant to saline and alkali, and can only grow on soils with a sodium chloride content of less than 0.3%. Excess Na + in soil will be plant Normal 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 higher model plant Arabidopsis to study the salt-tolerant molecular mechanism of plants, which has made breakthroughs 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.
  • the first aspect of the present invention provides a gene encoding an ion channel-like protein SOS1 of cotton having the sequence of 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 35S-GhS0S-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 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 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, 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 salt tolerance of a plant and for use in plant breeding Use;
  • the plant is tobacco.
  • a seventh aspect of the invention provides the amino acid sequence encoded by the gene of the first aspect of the invention, as set forth in SEQ ID NO: 1.
  • DRAWINGS Figure 1 is a construction flow of a plant expression vector (35S-GhS0S1-2300) of GhSOS1.
  • Figure 2 is a plasmid map of the plant expression vector (35S-GhS0S1-2300) of GhSOS1.
  • FIG 3 shows the results of salt tolerance simulation experiments of GhSOS1 transgenic T1 tobacco plants (right, T ⁇ W3) and non-transgenic tobacco plants (left) as controls.
  • Figure 4 shows the results of protein expression verification at the transcriptional level of GhSOS1 transgenic T1 tobacco plants and non-transgenic control plants.
  • M is Marker
  • 1_8 is a normal growing transgenic plant
  • 9_12 is a control plant
  • 13 is a positive control (GhSOSl).
  • BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples.
  • Example 1 Cotton SSH library construction under salt stress:
  • a subtractive library was constructed by inhibition subtractive hybridization using the method shown in Clontech's PCR-selectTM cDNA Subtraction Kit.
  • the mRNA of the roots of the cotton seedlings treated with NaCl for 6 hours was used as a tester during the experiment, and the mRNA of the roots of the untreated cotton seedlings was used as a driver.
  • African cotton (National Cotton Medium-Term Library, 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 medium (9 ⁇ 39 mM KN0 3 , 0. 625 mM KH2P0 4 , 10. 3 mM NH 4 N0 3 , 0.75 mM MgS0 4 , 1.
  • the test seedlings were divided into 2 groups of 4 plants each.
  • the first group was a control group, cultured at 25 ° C under light, and placed in 1/2 MS liquid medium.
  • the second group was treated with 25 ° C, light culture, placed in 1/2 MS liquid medium supplemented with a final concentration of 200 mM NaCl, and treated for 6 hours. After the treatment, the roots of the two groups were cut out in time. After rapid freezing with liquid nitrogen, it was stored in a -70 ° C refrigerator.
  • Clontech's PCR-selectTM cDNA Subtraction Kit Suppression subtractive hybridization was performed as indicated by Clontech's PCR-selectTM cDNA Subtraction Kit.
  • Clontech's PCR-Select cDNA Subtraction Kits were used to reverse-transcribe the mRNAs of Driver and Tester to obtain double-stranded cDNA, and then subtracted hybridization using 2 Tester and 2 g Driver cDNA as starting materials.
  • the Tester cDNA and Driver cDNA were digested with Rsal for 1.5 h in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated to the linker.
  • Tester cDNAs with different adaptors were mixed with an excess of Driver for the first subtractive hybridization.
  • the two first subtractive hybridization products were mixed, and then subjected to a second subtractive hybridization with the freshly denatured Driver cDNA, and the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.
  • the second PCR product of the forward subtracted hybrid cDNA fragment (QIAquick PCR Purification Kit, purchased from Qiagen) was ligated to the pGEM-T Easy (purchased from Promega kit) vector, according to the procedure of the pGEM-T Easy kit, specific steps 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, ⁇ 4 ligase buffer 5 ⁇ 1, pGEM-T Easy vector 1 ⁇ 1, T4 DNA ligase 1 ⁇ ⁇ was ligated overnight at 4 °C. 10 ⁇ L of the reaction product was added to 100 ⁇ L of competent E.
  • coli JMI09 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, and 250 ⁇ L of LB medium (1 % Tryptone was purchased from 0X0ID, 0.5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm. It was placed in a 37 °C water bath, shaken at 225 r/min for 30 min, and 200 ⁇ L of bacterial solution was planted in 50 ⁇ g. /mL ampicillin LB (ibid.) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA) was cultured on a plate at 37 °C for 18 h.
  • LB medium 1 % Tryptone was purchased from 0X0ID, 0.5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm. It was placed in a 37 °C water bath, shaken at 225 r/min for 30 min, and 200
  • sequence was SEQ ID No: 3. Sequence analysis indicated that the encoded amino acid sequence of the sequence belonged to the ion channel protein S0S1, and the full version of the clone Gh-S2-073 was encoded in this paper.
  • the long gene was named GhSOSl, and the corresponding protein of the gene was named S0S1.
  • GhSOSl 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 3' primer AUAP (provided with the kit), using mRNA reverse transcribed cDNA as a template.
  • the specific steps are as follows: Ex Taq purchased from TAKARA, 50 ⁇ 1 PCR reaction system: 5 ⁇ 1 ⁇ Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ 1 mRNA reverse transcribed cDNA, 1.0 ⁇ 1 Ex Taq, 10 ⁇ M Primers SEQ ID NO: 4 and AUAP each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50-fold with biguanide water and then 2.0 ⁇ l was used as a template.
  • the second round of PCR amplification was carried out with SEQ ID NO: 5 and the 3' primer AUAP.
  • the specific steps were as follows: 50 ⁇ 1 PCR reaction system: 5 ⁇ ⁇ ⁇ 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 AUAP 2.0 ⁇ l each And 35 ⁇ ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72V for 2 min, after 33 cycles, extension at 72 °C for 10 min.
  • the second PCR product recovered fragment was approximately 2400 bp band (Gel Extraction Kit was purchased from OMEGA) and ligated into pGEM_T Easy Vector, transformed into E. coli JM109 (specific method as above), and 10 white colonies were randomly picked up to contain 50 g/mL.
  • Ampicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20%, and stored at -80 ° C until use.
  • SEQ ID NO: 5 and 3' primers AUAP were subjected to bacterial cell PCR amplification, and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the 3' end of the cDNA of the gene was obtained.
  • GhSOSl GSP3 SEQ ID NO: 6:
  • GhSOSl GSP4 SEQ ID NO: 7:
  • GhSOSl GSP5 SEQ ID NO: 8: ACAGATTGCATCAACAGATTTG
  • 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 5' universal primer AAP (provided with the kit), and 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: Ex Taq was purchased from TAKARA, 50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2. 5 mM dNTP, 2. 0 ⁇ mRNA reverse transcribed cDNA, 1. 0 ⁇ Ex Taq, 10 ⁇ primer SEQ ID NO: 7 and AAP each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50 times with double distilled water, and then taken as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 8 and the 3' primer AUAP.
  • the specific steps were as follows: 50 ⁇ l ⁇ ? : 5 ⁇ ⁇ Buffer, 3 ⁇ 2. 5 mM dNTP, 2. 0 ⁇ diluted first round PCR product, 1.
  • the obtained 5 ' RACE product was cloned and sequenced, and spliced with the 3 ' RACE product sequencing result.
  • the cDNA sequence of GhSOS1 was obtained as SEQ ID NO: 9.
  • GhSOSIF SEQ ID NO: 10:
  • GhSOSIR SEQ ID NO: 11:
  • GhSOS1 The full length of GhSOS1 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 ⁇ ⁇ primer SEQ ID NO: 10 and SEQ ID NO: 11 each of 2.0 ⁇ l, and 30 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
  • the PCR amplification product was added with A tail: The PCR product was added with 2.5 times of absolute ethanol, placed at _20 ° C for 10 minutes, centrifuged, de-cleared, air-dried, and dissolved in 21 ⁇ ⁇ double distilled water. Add 2. 5 ⁇ ⁇ ⁇ ⁇ ⁇ Buffer, 0. 5 ⁇ 1 5 mM dATP, 2. 5 ⁇ 1 ⁇ ⁇ ⁇ Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes.
  • a DNA fragment of about 3300 bp was recovered (Omega recovery kit), ligated into the pGEM T-easy vector (GhSOS1-pGEM plasmid was obtained), transformed into JM109 (method as above), and 10 white colonies were randomly picked up to contain 50 g. /mL ampicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20%, and stored at -80 ° C until use.
  • SEQ ID NO: 10 and SEQ ID NO: 11 were subjected to bacterial cell PCR amplification (reaction system and reaction conditions are the same as above), and four positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the sequence was SEQ ID. N0: 2, the amino acid sequence of the encoded protein is SEQ ID NO: 1.
  • ATCGCTTCCA GGCATCTTCT TCGCGGCACC AGAGTTCCTT ACACTGTCGC TTTGCTCATC
  • 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. . 35S and Tnos were selected as promoters and terminators of the GhSOS1 gene.
  • Pnos was amplified using the primers SEQ ID NO: 12 and SEQ ID NO: 13 with the plant expression vector PBI 121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min.
  • pCAMBIA2300-1 was obtained by restriction enzyme cleavage to pCAMBIA2300 (promega, T4 ligase cassette) by EcoRI, Bglll.
  • SEQ ID NO: 14 and P SEQ ID NO: 15 Amplification of Tnos using PBI121 as a template, using TAKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min.
  • pCAMBIA2300-2 was obtained by cleavage of KpnI and EcoRI to pCAMBIA2300_l (promega T4 ligase cassette).
  • TCAGAATTCCCAGTGAATTCCCGATCTAGTA SEQ ID NO: 16 and P SEQ ID NO: 17 Amplification of the 35S promoter with pCAMBIA2300 as a template, using TAKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ 1 PCR reaction system: 10 ⁇ 1 5XPS Buffer, 3 ⁇ 1 2 ⁇ 5 mM dNTP, 1.0 ⁇ 1 pCambia 2300 plasmid, 1 ⁇ 0 ⁇ 1 PrimeSTAR, 10 ⁇ ⁇ primers SEQ ID NO: 16 and SEQ ID NO : 17 each of 2.0 ⁇ 1, and 31 ⁇ 1 of double distilled water.
  • SEQ ID NO: 18 and SEQ ID NO: 19 amplify GhSOS1 (template is obtained in Example 2)
  • GhSOSl-pGEM plasmid GhSOSl-pGEM plasmid
  • TaKaRa's PrimeSTAR HS DNA polymerase 50 ⁇ 1 PCR reaction system: 10 ⁇ 1 5 X PS Buffer, 3 ⁇ 1 2 ⁇ 5 mM dNTP, 1. 0 ⁇ 1 GhSOSl-pGEM, 1. 0 ⁇ 1 PrimeSTAR, 10 ⁇ ⁇ primer SEQ ID NO : 18 and SEQ ID NO: 19 each of 2.0 ⁇ l, and 31 ⁇ l of double distilled water.
  • Agrobacterium LBA4404 (available from Biovector Science Lab, Inc) Competent preparation: Agrobacterium LBA4404 was plated on LB solid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml streptomycin 1-2 days in advance Single spot inoculation, culture 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. Incubate overnight at 28 °C (about 12-16 h) to 0D600 value. 0. 4, forming a seed bacterial liquid.
  • Transformation of Agrobacterium Thaw competent cells on ice, add 1 ⁇ l of plasmid to 40 ⁇ l of competent cells, mix and ice bath for about 10 min. Transfer the mixture of competent and DNA to a pre-cooled electric shock cup with a gun and tap to bring the suspension to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from bio-rad) on the slide of the electric shock chamber and push the slide to place the electric shock cup on the base electrode of the electric shock chamber. When using a 0. lcm electric shock cup, the program of MicroPulser (purchased from bio-rad) is set to "Agr" and the electric shock is applied once.
  • the electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added. Quickly and gently spread the cells with a gun. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h. Take 100 ⁇ 200 ⁇ l of the bacterial solution and plate on the corresponding resistant screening medium (LB solid medium, containing 50 g/ml rifampicin, 50 yg/ml streptomycin, 50 ⁇ g/ml card) Natamycin), cultured at 28 °C.
  • LB solid medium containing 50 g/ml rifampicin, 50 yg/ml streptomycin, 50 ⁇ g/ml card
  • the leaves of sterile seedlings were cut into 5 mm ⁇ 5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing expression vector 35S-GhS0Sl-2300 in logarithmic growth phase for 10 min, and the bacterial culture was sucked up and co-cultured under dark conditions. 2 days (MS medium).
  • the leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (NAA) + 50 mg/L kanamycin + 500 mg/L cephalosporin).
  • the buds After culturing for about 45 days under light conditions, the buds should be grown and transferred to rooting medium (MS+50 mg/L kanamycin + 500 mg/L cephalosporin) for about 30 days, until the root system is developed. The seedlings were transferred to MS medium supplemented with 500 mg/L cephalosporin for number storage.
  • rooting medium MS+50 mg/L kanamycin + 500 mg/L cephalosporin
  • the obtained transgenic tobacco leaves were extracted and extracted with DNA (the Arabidopsis thaliana DNA extraction method in Example 3), using SEQ ID NO: 9: P SEQ ID NO: 10 (50 ⁇ l PCR reaction system: 5 ⁇ 1 10 X Ex Buffer) , 3 ⁇ 1 2 ⁇ 5 mM dNTP, 2.0 ⁇ 1 DNA, 1.0 ⁇ 1 Ex Taq, 10 ⁇ M primers SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 ⁇ 1, and 35 ⁇ 1 of double steaming PCR conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, 72V extension for 10 min), PCR identification, preservation Positive plants were numbered T.
  • W1_T. W20 Example 6 Salt Tolerance Simulation Experiment and Functional Identification of Overexpressing GhSOS1 Transgenic Tobacco T1
  • the sterilized vermiculite was permeated with 1/2 MS medium.
  • T. W1-T. W20 and control tobacco seeds were sown on vermiculite, 15 seeds per pot, 25 ° C, 14 hours light culture / 10 hours dark culture cycle, each 1/2 MS was poured once every 5 days, and after 25 days of culture, SEQ ID NO: 10 and P SEQ ID NO: 11 were subjected to PCR detection to remove negative plants. Select the same size of transgenic tobacco and control tobacco for salt tolerance experiment, and keep 4-5 seedlings of uniform size in each pot.
  • the transgenic tobacco, the control tobacco was poured with 1/2 MS of 200 mM NaCl, and the dark culture cycle was carried out at 25 ° C, 14 hours light culture/10 hours.
  • the salt tolerance of the T1 transgenic plants showed that the control plants could not grow normally, but T ⁇ W3, T ⁇ W5, T ⁇ W8, T ⁇ W12
  • the six strains of T ⁇ W16 and T ⁇ W19 can grow normally and show obvious salt tolerance (see Figure 2, taking T ⁇ W3 as an example, T ⁇ W5, T ⁇ W8, T ⁇ W12, The results of T ⁇ W16 and T ⁇ W19 are similar to T ⁇ W3, which is not shown here).
  • Example 7 Verification of SOS1 protein expression at the transcriptional level
  • T ⁇ W3, T ⁇ W5, T ⁇ W8, T ⁇ W12, T ⁇ W16, T ⁇ W19 normal growth T1 plants were randomly selected, and 6 control plants were randomly selected and extracted with plant RNA.
  • Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription kit Superscript III Reverse Transcriptase (1 g total RNA as a template, reverse transcription primer SEQ ID NO: 11).
  • GhSOS1 was detected by SEQ ID NO: 20 and SEQ ID NO: 21, and the relative expression of SOS1 protein was detected.
  • PCR was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and reverse transcribed 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 N0: 20 and SEQ ID NO: 21 each of 2.0 ⁇ l, and 30 ⁇ of double distilled water.
  • M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-8 is a transgenic plant, 9-12 is a control plant, and 13 is a positive control (GhSOSl, SEQ ID NO: 2).
  • the strip size shown in the figure is the same as the size of the positive control. The results showed that the normal growing transgenic plants had stronger transcription of GhSOS1 and the control plants were not transcribed.
  • SEQ ID NO: 20 TCTGATTCTTCTGTACATCTTTG
  • SEQ ID NO: 21 TTGAGTGCTTGTTTCGTATGCTC

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Abstract

L'invention concerne une protéine de canal ionique SOS1 dérivée de Gossypium spp. et le gène codant celle-ci. L'invention concerne également l'utilisation de ladite protéine pour la culture de plantes transgéniques à tolérance au sel augmentée.
PCT/CN2012/001426 2012-10-23 2012-10-23 Protéine de canal ionique du coton, gène codant et leurs utilisations Ceased WO2014063271A1 (fr)

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CN201280076371.7A CN105026565B (zh) 2012-10-23 2012-10-23 一个棉花离子通道类蛋白及其编码基因与应用
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