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WO2015024143A1 - Protéine à doigts de zinc zat10-1 provenant du coton, gène codant et applications associés - Google Patents

Protéine à doigts de zinc zat10-1 provenant du coton, gène codant et applications associés Download PDF

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Publication number
WO2015024143A1
WO2015024143A1 PCT/CN2013/000988 CN2013000988W WO2015024143A1 WO 2015024143 A1 WO2015024143 A1 WO 2015024143A1 CN 2013000988 W CN2013000988 W CN 2013000988W WO 2015024143 A1 WO2015024143 A1 WO 2015024143A1
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plant
seq
gene
expression vector
plants
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Chinese (zh)
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梁远金
崔洪志
王建胜
梁丽
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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 cotton-derived zinc finger protein ZAT10-1 and its encoding gene, and its use in the cultivation of transgenic plants having 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.
  • Higher plant cells can sense changes in physicochemical parameters in the external environment through various pathways, thereby transforming extracellular signals into intracellular signals, and finally transmitting stress signals to the nucleus through a series of signal transductions.
  • the transcription factor is activated, and the activated transcription factor acts on the functional gene to initiate the expression of the stress response gene to increase the tolerance of the plant.
  • the present inventors cloned a zinc finger protein (designated herein as ZAT10-1) encoding gene of Gossypiim herbaceim D of African cotton using SSH (suppression subtractive hybridization) in combination with RACE (rapid amplification of cDNA ends). And its DNA sequence was determined. Furthermore, it was found that after introduction into the recipient plant and expression thereof, the salt tolerance of the recipient plants was significantly improved, and these traits were stably inherited.
  • ZAT10-1 zinc finger protein
  • the first aspect of the invention provides a gene encoding a zinc finger protein ZAT10-1 of cotton (designated herein as
  • GhZA T10-l 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
  • An expression control sequence for the expression vector is operably linked; preferably, the vector is the 35S-GhZAT10-l-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 present 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 causing the gene Expression;
  • the plant is Arabidopsis thaliana.
  • a fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or a plant 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 Tissue;
  • 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.
  • a 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. 1 is a construction flow of a plant expression vector (35S-GhZAT10-l-2300) of a gene (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector of the gene (35S-GhZAT10-l-2300).
  • Figure 3 shows the results of salt tolerance simulation experiments of transgenic 1 ⁇ transgenic Arabidopsis plants (right panel, TVV3-6) and non-transgenic Arabidopsis plants (left panel, CK).
  • Figure 4 is a verification result of molecular detection of genes at the transcriptional level in 1 ⁇ generation transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR.
  • the upper and lower partial bands shown in the figure belong to ⁇ and tac - respectively as internal parameters.
  • 1_4 is a salt-tolerant non-transgenic control Arabidopsis plant
  • 5-12 is a salt-tolerant T1 transgenic Arabidopsis plant (in the order of ⁇ 3-1, ⁇ 3-6, ⁇ 3-8 and ⁇ ⁇ 3_16 four salt-tolerant strains, 2 strains per strain)
  • 13 is 35S-GhZAT10-l-2300 plasmid PCR positive control
  • 14-17 is transgenic salt-tolerant Arabidopsis plants (subordinate to 2 strains) 2 strains were tested, and 18 was a blank control without template.
  • the subtraction subtraction library was constructed by the method of inhibition subtractive hybridization using the method shown by Clontech's PCR-selectTM cDNA Subtraction Kit.
  • the mRNA of the leaves of the cotton seedlings treated with salt was used as a tester during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a control.
  • the specific steps are as follows:
  • African cotton (National Cotton Medium Term Bank, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) Seeded onto a sterilized vermiculite substrate, cultured at 25 ° C, light dark cycle 16 h / 8 h, each 1/2MS liquid medium (containing 9.39 mM KN0 3 , 0. 625 mM KH 2 P0 4 , 10. 3 mM N N0 3 , 0.75 mM MgS0 4 , 1. 5 mM CaCl 2 , 50 ⁇ M KI, 100 ⁇ ⁇ H 3 B0 3 , 100 ⁇ ⁇ MnS0 4 , 30 ⁇ M ZnS0 4 , 1 ⁇ M N3 ⁇ 4Mo0 4 , 0. 1 ⁇ ⁇ CoCl 2 , 100 ⁇ M N3 ⁇ 4 EDTA, 100 ⁇ M FeS0 4 ) Once. 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, and placed in 1/2 MS liquid medium.
  • the second group is the processing group, 25. C. Incubate under light, place in 1/2 MS liquid medium supplemented with a final concentration of 200 mM NaCl, and treat for 6 hours. After the treatment, the leaves of the two groups of seedlings should be cut out in time and quickly frozen with liquid nitrogen. Store in a -70 ° C refrigerator.
  • the cotton leaves of the control and the salt-treated group were each 0.5 g, and the total RNA of the cotton leaves was extracted with the plant RNA extraction kit (Invitrogen). Absorbance values of total RNA at 260 nm and 280 nm, 0D 26 , were determined using a HITACHI UV spectrophotometer U-2001. /0D 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 The integrity of the RNA is good. The mRNA was isolated using Qiagen's Oligotex mRNA purification assay [JC (Purification of poly A+ RNA from total RNA).
  • JC Qiagen's Oligotex mRNA purification assay
  • Suppression subtractive hybridization was performed as indicated by Clontech's PCR-selectTM cDNA Subtraction Kit kit.
  • the Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain a 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 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.
  • Two tester cDNAs with different adaptors were mixed with excess Driver cDNA for the first forward subtractive hybridization.
  • the products of the two first subtractive hybridizations were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and then the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.
  • the second PCR product of the combined positive subtractive hybridization cDNA fragment (using QIAquick PCR) according to the method described in the product manual for the pGEM-T Easy kit (purchased from Promega) Purification Kit purification, purchased from Qiagen) and pGEM_T Easy vector, the specific steps are as follows: The following components were sequentially added using a 200 ⁇ PCR tube: The second PCR product of the purified positive subtractive hybridization cDNA fragment 3 ⁇ 1, 2 ⁇ ⁇ 4 ligase buffer 5 ⁇ l, pGEM-T Easy vector 1 ⁇ l, T4 DNA ligase 1 ⁇ ⁇ , ligated overnight at 4 °C. Take 10 ligation reaction products, add 100 ⁇ L of E.
  • coli JM109 competent cells purchased from TAKARA
  • ice bath for 30 min heat shock for 60 s
  • ice bath for 2 min then add 250 ⁇ L LB medium (including 1 % Tryptone was purchased from 0X0ID, 0. 5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm.
  • LB medium including 1 % Tryptone was purchased from 0X0ID, 0. 5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm.
  • cultured at 225 r/min for 30 min cultured at 225 r/min for 30 min, and 200 ⁇ L of culture solution after shaking culture was taken.
  • Gh ZAT10-1_2 GSP2 SEQ ID NO: 5:
  • Reagents come with universal primers:
  • the experimental procedure was performed according to the kit instructions (3' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • SEQ ID NO: 4 and 3' primer SEQ ID NO: 7 (the AUAP primer provided by the kit), reverse transcription of SEQ ID NO: 6 primer (AP primer derived from the kit) and cotton mRNA.
  • the cDNA was used as a template for the first round of PCR amplification. Specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 ⁇ Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ 1 mRNA reverse transcribed cDNA, 1.0 ⁇ 1 Ex Taq (purchased from TAKARA), 10 ⁇ M primer SEQ ID NO : 4 and P SEQ ID NO: 7 each of 2.0 ⁇ 1, and 35 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50 times with double distilled water and 2.0 ⁇ l was used as a template, and SEQ ID NO:
  • PCR reaction system 5 ⁇ 1 ⁇ Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ l diluted first round PCR product, 1.0 ⁇ 1 Ex Taq, 10 ⁇ primers SEQ ID NO: 5 and SEQ ID NO: 7 each of 2.0 ⁇ 1, and 35 ⁇ 1 of double distilled water.
  • PCR reaction conditions 94 ° C pre-denaturation 5 Min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the second PCR product recovery fragment (Gel Extraction Kit was purchased from OMEGA) was ligated to pGEM-T Easy Vector, transformed into E. coli JM109 (the same method as above), and 8 white colonies were randomly picked up to contain 50 g/mL ampicillin. Incubate in LB liquid medium, incubate at 37 °C overnight, add glycerol to a final concentration of 20%, and store at -80 °C until use. SEQ ID NO: 5 and 3' primer SEQ ID NO: 7 were used for PCR amplification of bacterial cells, and 6 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Co., Ltd. for sequencing and sequencing, and 3' cDNA of the gene was obtained. end.
  • GhZATIO- 1_1 GSP1 SEQ ID NO: 9:
  • GhZATIO- 1_2 GSP2 SEQ ID NO: 10:
  • GhZATIO- 1_3 GSP SEQ ID NO: 11:
  • 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 (reverse transcription primer SEQ ID NO: 9) was used as a template for the first round of PCR amplification.
  • the specific steps are as follows: 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 (purchased from TAKARA), 10 ⁇ M primers SEQ ID NO: 10 and AAP each 2. 0 ⁇ 1, and 35 ⁇ 1 Double steamed water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50-fold with double distilled water, and then 2.0 ⁇ l was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 11 and the universal primer AUAP (provided by the kit), and the specific steps were as follows: 50 ⁇ 1 PCR reaction system: 5 ⁇ 1 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 1 2. 5 mM dNTP, 2. 0 ⁇ 1 diluted first round PCR product, 1. 0 ⁇ 1 Ex Taq, 10 ⁇ M Primers SEQ ID NO: 11 and AUAP were each 2.0 ⁇ l, and 35 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 V for 2 min), extension at 72 °C for 10 min.
  • the second round of PCR product was recovered (purchased from OMEGA using the Gel Extraction Kit), ligated into the pGEM_T Easy vector, transformed into JM109 competent cells (specific method as above), and the transformed bacterial solution was coated in 50 Screening was performed on LB solid medium of yg/mL ampicillin, 40 ⁇ g/mL X_gal, 24 ⁇ g/mL IPTG.
  • the sequence obtained by cloning the obtained 5' RACE product was spliced with the sequence of SEQ ID NO: 8, and SEQ ID NO: 12 was obtained.
  • GhZA T10-l F SEQ ID NO: 13:
  • GhZA T10-l R SEQ ID NO: 14:
  • the GhZA TW-1 full-length coding sequence was cloned by SEQ ID NO: 13 and SEQ ID NO: 14.
  • 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 HS DNA polymerase, 10 ⁇ ⁇ primer SEQ ID NO: 13 and SEQ ID NO: 14 each of 2.0 ⁇ l, and 30 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • PCR amplification product plus A tail PCR product plus 2.5 times the volume of absolute ethanol, placed at _20 °C for 10 minutes, centrifuged, supernatant, dried, dissolved in 21 ⁇ l of double distilled water. Add 2. 5 ⁇ 1 ⁇ ⁇ ⁇ 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 900 bp was recovered (using the Omega recovery kit) and ligated into the pGEM T-easy vector (GhZA TW+pG was obtained, and the resulting plasmid was transformed into JM109 competent cells (method supra)
  • 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. Inoculated in LB liquid medium containing 50 ⁇ g/mL ampicillin, cultured overnight at 37 °C Glycerol is brought to a final concentration of 20% and stored at -80 °C until use.
  • SEQ ID NO: 13 and SEQ ID NO: 14 were tested for bacterial liquid PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • the sequence is SEQ. ID NO: 2, the amino acid sequence of the encoded ZAT1O-1 protein is shown in 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 constitutive promoter 35S containing the double enhancer and the terminator Tnos were selected as promoters and terminators of the gene, respectively.
  • the construction process is shown in Figure 1.
  • Primer SEQ ID NO: 15 and SEQ ID NO: 16 were used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ 1 PCR reaction system: 10 ⁇ ⁇ 5XPS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 ⁇ 121, 1 ⁇ 0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ M primer SEQ ID NO: 15 and SEQ ID NO: 16 each of 2.0 ⁇ 1, and 31 ⁇ l of double distilled water.
  • PCR reaction conditions pre-variability at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was digested with f coR I, Bgl II and ligated into pCAMBIA2300 (purchased from Promega, T4 ligase cassette) to obtain pCAMBIA2300_1.
  • Primers SEQ ID NO: 17 and SEQ ID NO: 18 were used to amplify Tnos using the pBI121 plasmid as a template, using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was digested with Sac I, EcoR I and ligated into pCAMBIA2300-1 to obtain pCAMBIA2300_2.
  • SEQ ID NO: 17 AAGGAGCTCGAATTTCCCCGATCGTTCAAA
  • SEQ ID NO: 18 AAGGAGCTCGAATTTCCCCGATCGTTCAAA
  • the CaMV 35S promoter was amplified using the primers SEQ ID NO: 19 and SEQ ID NO: 20 using the pCAMBIA2300 plasmid as a template.
  • PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 ⁇ 1 PCR reaction system: 10 ⁇ ⁇ 5XPS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 pCAMBIA 2300 plasmid DNA, 1.0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ primer SEQ ID NO: 19 and SEQ ID NO: 20 each of 2.0 ⁇ 1, and 31 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the obtained PCR product was ligated by the digestion of ⁇ ⁇ ' ⁇ ! III, Pst I (the ligation method is the same as above) to pCAMBIA2300-2 to obtain pCAMBIA2300_3.
  • Primers SEQ ID NO: 21 and P SEQ ID NO: 22 ⁇ GhZATlO-1 template is the positive GhZAT1O+G plasmid obtained in Example 2), using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated by / 3 ⁇ 4 ⁇ I, Sac I (ligation as described above) to pCAMBIA2300-3, and the plant expression vector 35S-GhZAT10-l-2300 (Fig. 2) was obtained.
  • Agrobacterium tumefaciens 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. Incubate at 28 °C for 1 to 2 days. Pick a single colony and inoculate 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.
  • the glycerin was repeatedly washed 3-4 times; the bacterial pellet was resuspended by adding an appropriate amount of ice-cold 10% glycerol to prepare LBA4404 competent cells. It was then dispensed in 40 ⁇ l/tube and stored at _70 °C for later use.
  • Transformation of Agrobacterium Thaw competent cells on ice, add 1 ⁇ l of the positive 35S-GhAZF2-l-2300 plasmid obtained in Example 3 to 40 ⁇ l of competent cells, mix and heat for about 10 min. .
  • the mixture of the competent cells and the 35S-GhAZF2-l-2300 plasmid after the ice bath was transferred to an ice-cold 0.1 cm-sized electric shock cup (purchased from bio-rad) using a micropipette. Knock the suspension to the bottom, taking care not to have air bubbles.
  • the electric shock cup is placed on the slide of the electric shock chamber, and the slide is pushed to place the electric shock cup to the base electrode of the electric shock chamber.
  • the bacterial solution of the Agrobacterium LBA4404-transformed positive clone containing the 35S-GhAZF2-l-2300 plasmid obtained in Example 4 was inoculated to an LB liquid medium containing 50 ⁇ g/ml kanamycin overnight, and the next morning.
  • the cells were inoculated at 1:50 into a new LB medium (1 L) containing 50 ⁇ g/ml kanamycin and cultured for about 8 hours to Agrobacterium liquid 0D 6 . . Between 1. 0 and 1.2.
  • Seed disinfection first soak for 10 minutes with 70% ethanol, 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 1/2 MS solid screening medium containing 50 ⁇ g/ml kanamycin (a maximum of 1500 seeds were plated in a 150 mm diameter plate), and vernalized at 4 °C. day. Then, the cells were cultured for 7-10 days at a constant temperature of 22 ° C, an illumination intensity of 3500-4000 lx, and an illumination period of 8 h dark/16 h. Seeds that can germinate and grow normally are transgenic seeds.
  • Will T. A3-1 to T. A3-20 was planted in 2 pots per transformation line, 2 pots were planted in non-transgenic control Arabidopsis, and 20-30 seeds were seeded per pot. After sowing, the film is covered with a film to provide a moist environment for plant growth.
  • the temperature was 22 ° C
  • the light intensity was 3500-4000 lx
  • the photoperiod was 12 h dark / 12 h light culture
  • 1/2 MS liquid medium was watered every 7 days.
  • the leaves of the transgenic Arabidopsis lines were excised and their genomic DNA was extracted as a template, and PCR detection was carried out using primers SEQ ID NO: 13 and SEQ ID NO: 14.
  • Example 9 Salt tolerance test of overexpressing GhZA TW-1 transgenic Arabidopsis thaliana 1 ⁇ generation plants
  • Example 8 the transgenic Arabidopsis thaliana and the control Arabidopsis thaliana were each treated without treatment, and the 1/2 MS liquid medium was normally watered; in addition, each of the transgenic Arabidopsis thaliana and the control Arabidopsis thaliana was watered with 150 mM NaCl. 1/2 MS liquid medium, constant temperature 22 ° C, light intensity 3500-4000 lx, 12 hours light culture / 12 hours dark culture cycle, observe the experimental results after 10 days. Identification of salt tolerance of 1 ⁇ generation transgenic plants (T.
  • Example 8 transgenic 1 ⁇ generation plants with good salt tolerance in Example 9 were randomly selected (in turn, the above four salt-tolerant strains of ⁇ 3-1, ⁇ 3-6, ⁇ 3-8 and T ⁇ A3_16, each 2 strains), the control plants in Example 6 were randomly selected from 4 plants, each of which was cut with 150 mM NaCl for 14 days, 0. 05 g, and total RNA was extracted with a plant RNA extraction kit (invi trogen). The absorbance values of total RNA 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 Invi trogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (1 total RNA as a template and reverse transcription primer as SEQ ID NO: 14).
  • Atactin-2 http://www. uniprot.org/uniprot/Q96292
  • the PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTARHS DNA polymerase.
  • PCR reaction system 10 ⁇ 15 X PS Buf f er, 3 ⁇ 1 2 ⁇ 5 mM dNTP, 2.0 ⁇ 1 cDNA, 1.0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ M primer SEQ ID NO: 23 and SEQ ID NO: 24 each of 2.0 ⁇ l, and 30 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 32 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • ZAT10-1 protein The relative expression of ZAT10-1 protein was detected by amplifying GhZATW-1 by the primers of SEQ ID NO: 13 and SEQ ID NO: 14.
  • PCR reaction 50 yl PCR reaction system: 10 ⁇ 15 XPS Buffer, 3 ⁇ 12.5 mM dNTP, 2.0 ⁇ 1 cDNA, 1 ⁇ 0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ M primers SEQ ID NO: 13 and SEQ ID NO: 14 each of 2.0 ⁇ 1, and 30 ⁇ 1 of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 32 cycles (denaturation at 94 ° C for 30 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 1 min), extension at 72 ° C for 10 min.
  • the product electrophoresis results are shown in Figure 4.
  • the upper and lower partial strips shown in the figure belong to GhZAT10-l Atactin-2, respectively, with ⁇ iacii?- ⁇ as the internal reference.
  • . 1_4 is a non-tolerant non-transgenic control Arabidopsis plant
  • 5-12 is a salt-tolerant T1 transgenic Arabidopsis plant (in turn, ⁇ 3-1, T ⁇ A3-6, TVV3-8, and ⁇ 3 -16 four salt-tolerant strains, 2 strains per strain)
  • 13 is a 35S-GhZAT10-l-2300 plasmid PCR positive control
  • 14-17 is a transgenic salt-tolerant Arabidopsis plant (subordinate to 2 strains) Department, 2 tests each)
  • 18 is a blank control without template.

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Abstract

La présente invention concerne une protéine végétale, un gène codant et une application associés, et en particulier une protéine à doigts de zinc ZAT10-1 provenant du coton, un gène codant associé, et une application dans la sélection d'une plante transgénique présentant une tolérance au sel améliorée.
PCT/CN2013/000988 2013-08-22 2013-08-22 Protéine à doigts de zinc zat10-1 provenant du coton, gène codant et applications associés Ceased WO2015024143A1 (fr)

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CN108866082B (zh) * 2018-08-06 2021-09-14 南京农业大学 大豆STF-3转录因子编码基因GmSTF-3及其应用
CN113717265B (zh) * 2021-09-08 2022-06-07 兰州大学 一种提高植物非生物胁迫耐受性的基因PalZAT10-1及其应用
CN120060275B (zh) * 2025-01-26 2025-11-04 中国农业大学 GhZAT10基因在促进植物耐低钾胁迫能力中的应用

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CN1818065A (zh) * 2005-10-14 2006-08-16 山东农业大学 棉花GhZFP1基因序列及其克隆与应用
CN101078015A (zh) * 2006-05-25 2007-11-28 中国科学院遗传与发育生物学研究所 一种柠条转录因子CkAREB及其在抗逆植物培育中的应用
CN101591383A (zh) * 2008-05-27 2009-12-02 中国农业科学院作物科学研究所 一种植物耐逆性相关蛋白及其编码基因与应用
CN102060919A (zh) * 2009-07-06 2011-05-18 中国农业科学院生物技术研究所 三个棉花abf/areb/abi5/dpbf类转录因子及其编码基因与应用

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CN1818065A (zh) * 2005-10-14 2006-08-16 山东农业大学 棉花GhZFP1基因序列及其克隆与应用
CN101078015A (zh) * 2006-05-25 2007-11-28 中国科学院遗传与发育生物学研究所 一种柠条转录因子CkAREB及其在抗逆植物培育中的应用
CN101591383A (zh) * 2008-05-27 2009-12-02 中国农业科学院作物科学研究所 一种植物耐逆性相关蛋白及其编码基因与应用
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