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WO2015042747A1 - Protéine déhydrine dh4 issue de thellungiella halophila, gène codant pour celle-ci, et utilisation du gène - Google Patents

Protéine déhydrine dh4 issue de thellungiella halophila, gène codant pour celle-ci, et utilisation du gène Download PDF

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WO2015042747A1
WO2015042747A1 PCT/CN2013/001172 CN2013001172W WO2015042747A1 WO 2015042747 A1 WO2015042747 A1 WO 2015042747A1 CN 2013001172 W CN2013001172 W CN 2013001172W WO 2015042747 A1 WO2015042747 A1 WO 2015042747A1
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gene
seq
plant
expression vector
plants
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陈文华
孙超
崔洪志
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • 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

Definitions

  • the present invention relates to plant proteins and coding genes thereof and applications thereof, and in particular to a dehydrin protein derived from small salt mustard/3 ⁇ 4 and its coding gene, and It is used in the cultivation of transgenic plants with improved drought tolerance. Background technique
  • the present inventors cloned a dehydrin protein of a small salt mustard using SSH (Suppression Subtractive Hybridization) in combination with RACE (Rapid Amplification of cDNA Ends) (designated as a gene encoding DH4, and the DNA thereof was determined. Sequence and found that when introduced into plants, the drought tolerance of transgenic plants can be significantly improved, and these Traits can be stably inherited.
  • the first aspect of the present invention provides a gene encoding a dehydrin protein DH4 of small salt mustard (herein named ThDH4) 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-7)H ⁇ -2300 vector shown in Fig. 2.
  • the 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 drought 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 drought tolerance of a plant and for use in plant breeding Use;
  • the plant is Arabidopsis thaliana.
  • the seventh aspect of the present invention provides the gene-encoded protein according to the first aspect of the present invention, which has an amino acid sequence as shown in SEQ ID NO: 1.
  • Fig. 1 is a construction flow of a plant expression vector C35S-7)H ⁇ -2300) of T DH4 (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector C35S-7)H ⁇ -2300) of T DH4.
  • FIG. 3 shows the results of drought tolerance simulation experiments of 7) H ⁇ T1 transgenic Arabidopsis plants (in the figure, T1D2) and non-transgenic Arabidopsis plants (in the figure, CK) as controls.
  • Fig. 3a is an Arabidopsis plant that grows normally for 20 days
  • Fig. 3b shows an Arabidopsis plant that has been treated for 14 days after normal growth for 14 days).
  • FIG. 4 is a graph showing the results of protein expression verification at the transcriptional level of transgenic T1 Arabidopsis plants and non-transgenic control plants.
  • M is DNA Ladder Marker (DL2000, TakaRa)
  • 1-5 is a drought-tolerant transgenic Arabidopsis T1 plant (in order: T1D1, T1D2, T1D3, T1D4, T1D5)
  • 6-10 is a drought-tolerant transgenic Arabidopsis thaliana T1 plants
  • 11-14 are non-transgenic Arabidopsis controls.
  • BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.
  • a subtractive 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 small salt mustard seedlings which were drought-treated during the growth period was used as a tester, and the mRNA of the leaves of the untreated small salt mustard seedlings was used as a driver. Specific steps are as follows:
  • Small salt mustard (T ellungiella halophila, purchased from the Yanlan Plant Breeding Center of Ulan Buh and Desert Green Botanical Garden, Bayannaoer City, Inner Mongolia, China), seeded on sterilized vermiculite, at 25 ° C, photoperiod 16 hours light / Incubate in 8 hours dark (light intensity 2000-3000 Lx) and pour 1/2MS medium per week ( 9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM NH 4 N0 3 , 0.75 mM MgS0 4 , 1.5 mM CaCl 2 , 50 ⁇ KI, 100 ⁇ ⁇ 3 ⁇ 3 , 100 M MnSO 4 , 30 ⁇ ZnS0 4 , 1 ⁇ ⁇ 2 ⁇ 0 4 , 0.1 ⁇ CoCl 2 , 100 ⁇ Na 2 EDTA, 100 M FeSO 4 ). When the seedlings were cultured for about 1 month, they were used for experiments.
  • the test seedlings were divided into two groups, each with 4 pots and 1 pot per pot.
  • the first group was a control group, which was cultured at 25 ° C, light cycle 16 hours light / 8 hours dark, and was normally watered.
  • the second group was the drought treatment group, cultured at 25 °C, photoperiod of 16 hours light/8 hours dark, stopped watering, and treated for 10 days. After the treatment, the leaves of the two seedlings were cut out in time and quickly frozen with liquid nitrogen. After storage in a refrigerator at -70 °C.
  • the leaves of the small salt mustard leaves of the control group and the drought treatment group were taken 0.1 g, and the total RNA of the leaves of the small salt mustard was extracted with a plant RNA extraction kit (purchased from Invitrogen).
  • the absorbance of total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001.
  • the OD 260 / OD 280 ratio was 1.8-2.0, indicating a higher total RNA purity; 1.0% agarose gel Electrophoretic detection of total RNA integrity, brightness of 28S bands It is about twice as large as the 18S band, indicating good RNA integrity.
  • mRNA was isolated using Qiagen's Oligotex mRNA Purification Kit (purification of poly A+ RNA from total RNA, purified polyA+ RNA from total RNA).
  • this experiment In order to increase the availability of the Expressed sequence tag (EST) (unigene), avoid the gene-free cleavage site and the obtained sequence in the untranslated region, this experiment simultaneously uses the endonuclease Haelll to tester cDNA according to the above steps.
  • the cDNA was digested with Driver cDNA and subjected to two forward subtractive hybridizations and two inhibitory PCR amplifications. Finally, the second inhibitory PCR products of the two groups of forward subtractive hybridization cDNA fragments were combined.
  • EST Expressed sequence tag
  • the second PCR product of the combined forward subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) and pGEM-T Easy vector according to the instructions of the pGEM-T Easy kit (purchased from Promega)
  • the specific steps are as follows: The following components are sequentially added to the 200 ⁇ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 ⁇ 1, 2 ⁇ 4 ligase buffer 5 ⁇ 1, pGEM-T Easy vector 1 ⁇ 1, T4 DNA ligase 1 ⁇ l, and ligated overnight at 4 °C.
  • reaction products were added and added to 100 ⁇ L of competent Escherichia coli JM109 (purchased from TAKARA), ice bath for 30 min, heat shock at 42 °C for 60 s, ice bath for 2 min, and 250 ⁇ L of LB liquid medium ( Containing 1% tryptone (Tryptone, purchased from OXOID), 0.5% yeast extract (Yeast Extract, purchased from OXOID) and P 1% NaCl (purchased from Sinopharm)) placed in a 37 ° C shaker at 225 r/ The culture was shaken for 30 min, and then 200 ⁇ of the bacterial solution was applied to 50 g/ml ampicillin, 40 g/mL X-gal, 24 g/mL IPTG (X-gal (5-bromo-4-chloro-3).
  • the cultured colony clones were subjected to nested PCR (primer Primer 1 and Primer 2R, PCR-selectTM cDNA Subtraction Kit from Clontech) for PCR amplification, and 166 positive clones were obtained, and then all were positive.
  • the clone was sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • SEQ ID NO: 3 is the 3' end sequence of the coding gene 7) H ⁇ . Based on the sequence of SEQ ID NO: 3 which has been obtained, the following three specific primers were designed as specific primers for reverse transcription primers and 5 'RACE.
  • TGTTGCCAATCGCTGAAGAAG YLS-113GSP3 SEQ ID NO:6: GCTCGGAGGACGAGGGACTAG
  • the kit comes with universal primers:
  • AAP SEQ ID NO: 7:
  • the GGCCACGCGTCGACTAGTAC experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • YLS-113GSP1 (SEQ ID NO: 4) was used as a reverse transcription primer, and the mRNA extracted from the leaves of the drought-treated group was used as a template for reverse transcription to obtain a cDNA template, and then according to the above 5' RACE kit instructions.
  • the first step of PCR amplification was carried out by adding the PolyC tail, and the product after tailing was used as a template.
  • the primer used was SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (the kit is self-contained, and I is modified by hypoxanthine. , c, g or t), the specific steps are as follows:
  • PCR reaction system 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ Ex Taq (purchased from TAKARA), 10 ⁇ primers SEQ ID NO: 4 and SEQ ID NO: 7 each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 45 s, annealing at 62 °C for 45 s, extension at 72 °C for 45 s), extension at 72 °C for 10 min.
  • 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 using SEQ ID NO: 5 and the general primer SEQ ID NO: 8.
  • the specific steps are as follows:
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • a strip of about 290 bp in the second PCR product (Golecular Extraction from OMEGA) was recovered and ligated into the pGEM-T Easy Vector vector, which was then transformed into JM109 (the method is the same as above), and 10 randomly picked.
  • White colonies were inoculated separately in LB liquid medium containing 50 g/mL ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C until use.
  • the primers SEQ ID NO: 5 and the 3' primer SEQ ID NO: 6 were used for PCR amplification (reaction system and reaction conditions as above). Three positive clones (01, 03 and 06) were obtained, and sent to the UK. Base (Shanghai) Trading Co., Ltd. sequenced to obtain a 5' end sequence of the cDNA of the gene.
  • the resulting 5' RACE product clone 06 was sequenced to obtain the sequence of SEQ ID NO: 9: 1 GGGGGGGGGG ATGGAGTCTA ATTATCAAAA CCAATCGGGA GCGCAGCAGA GTCACCAACA
  • SEQ ID NO: 10 is the full length sequence of 73 ⁇ 4 DH ⁇ . According to SEQ ID NO:
  • ThDH4F SEQ ID NO: 1 1:
  • ThDH4R SEQ ID NO: 12:
  • ThDH4 full-length coding sequence was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
  • RNA of the salt-treated mustard was extracted as a template, the primer SEQ ID NO: 13 was used as the reverse transcription primer, and the cDNA of the small salt mustard was obtained by reverse transcription, and then the PfuUltra II Fusion HS DNA Polymerase of stratagene was used to obtain the small amount obtained above.
  • the cDNA of the salt mustard was used as a template for PCR reaction.
  • PCR reaction system 5 ⁇ 10 X PfuUltra II reaction Buffer 0.5 ⁇ 25 mM dNTP, 2.0 ⁇ cDNA 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase, 10 ⁇ primers SEQ ID NO: 1 1 and SEQ ID NO: 12 2.0 ⁇ 1, and 37.5 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 95 °C for 2 min, 35 cycles (denaturation at 95 °C for 25 s, annealing at 51 °C for 25 s, extension at 72 °C for 30 min), extension at 72 °C for 5 min.
  • PCR amplification product plus A tail PCR product hydration to 400 ⁇ 1, first remove the protein with chloroform extraction, add the supernatant to the 3 ⁇ sodium acetate solution 40 ⁇ 1, add 2 volumes of absolute ethanol, -20 ° C for 10 minutes, Centrifuge, remove the supernatant, allow to dry, and dissolve in 21 ⁇ l of double distilled water. Add 2.5 ⁇ ⁇ ⁇ ⁇ ⁇ Buffer 0.5 ⁇ 5 mM dATP and 1.0 ⁇ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes.
  • Approximately 380 bp DNA fragment will be obtained Recovered (Omega recovery kit), ligated into pGEM T-easy vector (obtained ThDH4-pGEM plasmid), then transformed into JM109, randomly picked 8 white colonies and inoculated separately into LB liquid medium containing 50 g/mL ampicillin Medium culture, culture at 37 ° C overnight, add glycerol to a final concentration of glycerol 20% (volume ratio), and store at -80 ° C for later use.
  • the primers SEQ ID NO: 1 1 and SEQ ID NO: 12 were used for PCR amplification (reaction system and reaction conditions as above), and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, sequence.
  • SEQ ID NO: 2 the amino acid sequence of the encoded protein is SEQ ID NO: 1
  • Nucleotide sequence of the 73 ⁇ 4DH ⁇ coding gene SEQ ID NO: 2
  • the plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as the plant expression vector, and the Pnos promoter was used to replace the CaMV35S promoter containing the double enhancer in the ⁇ gene to reduce the expression of prion protein in plants. .
  • the 35S promoter and the terminator Tnos were inserted upstream of the Pnos promoter as the promoter and terminator of the ThDH4 gene, respectively, and the 73 ⁇ 4DH ⁇ gene was between the 35S promoter and the Tnos terminator.
  • Pnos was amplified using the primers SEQ ID NO: 14 and SEQ ID NO: 15 with the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ l ⁇ Reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ ⁇ 121 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 14 and SEQ ID NO: 15 each 2.0 ⁇ l, and 31 ⁇ 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 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 EcoRI and Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase cassette) to obtain pCAMBIA2300-1.
  • ATCCAGATCTAGATCCGGTGCAGATTATTTG The primers SEQ ID NO: 16 and SEQ ID NO: 17 were used to amplify Tnos using pBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ PCR reaction system: 10 ⁇ 5 xPS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ pBI121, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 ⁇ l, and 31 ⁇ 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 resulting PCR product was digested with Sacl, EcoRI and ligated into pCAMBIA2300-1 (Promega T4 ligase cassette) to obtain pCAMBIA2300-2.
  • the 35S promoter was amplified using the primers SEQ ID NO: 18 and SEQ ID NO: 19 using the pCAMBIA2300 plasmid as a template.
  • PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 ⁇ ⁇ Reaction system: 10 ⁇ 5 xPS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ diluted 50-fold pCAMBIA2300 plasmid, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 primer 8 £0 10 ⁇ 0: 18 and 8 £ 0 10 ⁇ 0: 19 each of 2.0 ⁇ , and 31 ⁇ 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 50 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated by HindIII and Pstl (connection method as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3 SEQ ID NO: 18:
  • TGACJGC ⁇ GAGAGATAGATTTGTAGAGAGAC ThDH4 was amplified using primers SEQ ID NO: 20 and SEQ ID NO: 21 (template was the positive 73 ⁇ 4 DH ⁇ -pGEM plasmid obtained in Example 2) using strafine Pfu Ultra II Fusion HS DNA Polymerase.
  • PCR reaction system 5 ⁇ lOxPfuUltra II reaction Buffer, 0.5 ⁇ l 25 mM dNTP, 2.0 ⁇ ThDH4-pGEM plasmid, 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase 10 ⁇ primers SEQ ID NO: 20 and SEQ ID NO: 21 Each of 2.0 ⁇ l, and 37.5 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 95 °C for 2 min, 35 cycles (denaturation at 95 °C for 25 s, annealing at 51 °C for 25 s, extension at 72 °C for 30 s), extension at 72 °C for 5 min.
  • the resulting PCR product was ligated by Pstl and Sacl (connection method as above) to pCAMBIA2300-3 to obtain a plant expression vector 35S-73 ⁇ 4DH ⁇ -2300.
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: 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 ⁇ ⁇ / ⁇ 1 rifampicin and 50 ⁇ ⁇ / ⁇ 1 streptomycin, and cultured overnight (about 12-16 hours) to OD 6 at 28 °C. The K) value is 0.4, and a seed bacterial liquid is formed.
  • Transformation of Agrobacterium The competent cells were thawed on ice, and 1 ⁇ M of the positive 35S-7)H ⁇ -2300 plasmid obtained in Example 3 was added to 40 ⁇ of the competent cells, and the mixture was mixed and ice bathed for about 10 min. Transfer the mixture of competent cells and 35S-ThDH4-2300 plasmid DNA to a ice-cold electric shock cup (purchased from bio-rad) with a pipette, tap to bring the suspension to the bottom of the electric shock cup, be careful not to have bubble. Place the electric shock cup 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.
  • a ice-cold electric shock cup purchased from bio-rad
  • the MicroPulser purchased from bio-rad
  • the MicroPulser is set to "Agr” and the shock is applied once.
  • the electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added.
  • the suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 hour with shaking.
  • Plants to be transformed Arabidopsis seeds (Columbia type, Arabidopsis thaliana Bioresource Center, Ohio State University) Seeded in peat soil, treated at 4 ° C for 3 days, placed at 23 ° C, 16 hours light Sprouting in an 8 hour dark incubator. After 7-10 days, transplanted into a plastic pot with a diameter of 7.5 cm containing peat and vermiculite (3:1 by volume), 6 plants per pot, placed at 23 ° C, 16 hours light / 8 hours dark Growing in the incubator.
  • Agrobacterium After removing the bacterial solution of the Agrobacterium-positive transformed clones preserved in Example 4, a single Agrobacterium colony was picked and inoculated into 10 mL of sterile LB liquid medium (containing 75 mg/L of rifampicin, 100 mg/L streptomycin and 100 mg/L kanamycin were shaken overnight at 250 °C/min at 28 °C. Then inoculate the obtained bacterial solution in a volume ratio of 1% to 2% to 200 mL of sterile LB liquid medium (containing 75 mg/L rifampicin, 100 mg/L streptomycin, and 100 mg/L Kana).
  • Inflorescence dip dyeing The above Agrobacterium-containing dyeing medium was added to a large-mouth container, and 200-300 mL of the Agrobacterium-containing dyeing medium was added to each container having a diameter of 9 cm for dip dyeing. Invert the plants so that all the above ground tissues are immersed in the Agrobacterium suspension for 3-5 s and gently agitate. There should be a liquid film on the plant after infiltration. Dip-infected plants are placed in plastic trays, covered with a clean plastic or cling film to moisturize, and then placed in low light or dark places overnight, taking care to prevent direct sunlight from the plants. Remove the cover approximately 12-24 hours after processing. The plants are cultured normally, and the plants are further grown for 3-5 weeks until the pods are browned and dried. The seeds were harvested and the seeds were dried and stored at 4 °C in a centrifuge tube.
  • Transgenic seed screening formulated with 1/4 MS (4.695 mM KN0 3 , 0.3125 mM KH 2 P0 4 , 5.15 mM H4NO3, 0.375 mM MgS0 4 , 0.75 mM CaCl 2 , 25 ⁇ ⁇ , 50 ⁇ ⁇ 3 ⁇ 3 , 50 M MnSO 4 , 15 M ZnS0 4 , 0.5 ⁇ ⁇ 2 ⁇ 0 4 , 0.05 M CoCl 2 , 50 ⁇ Na 2 EDTA , 50 M FeSO4)
  • a large amount of elemental aqueous solution add 0.8% agar powder, heat in a microwave oven until the agar is completely dissolved, and then cool to about 50 ° C, add the required amount of 50 mg of kanamycin, shake well After pouring 25 mL into each dish, the experiment bench can be sown after cooling and solidification.
  • SEQ ID NO: 11 P SEQ ID NO: 12, 50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ 1 ⁇ ⁇ 10 ⁇ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 45 s, annealing at 51 °C for 45 s, extension at 72 °C for 45 s), extension at 72 °C for 7 min, identification of PCR as positive
  • the plants were numbered (T1D1-T1D12) and saved.
  • Example 6 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis thaliana T1 plants overexpressing ThDH4 The sterilized vermiculite was soaked with 1/2 MS medium.
  • T1D1-T1D6 and control Arabidopsis seeds were planted on vermiculite, 10 seeds per pot, 25 °C, 10 hours light culture / 14 hours dark culture cycle, 1/2MS every 7 days, after 20 days of culture 4-5 seedlings of uniform size were kept in each pot for drought experiments.
  • the drought resistance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that the control plants were wilting, while T1D1, T1D2, T1D3, T1D4, T1D5 and T1D6 had 24 strains (each strain).
  • PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template.
  • 50 ⁇ l ⁇ Reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primer SEQ ID NO: 11 and P SEQ ID NO: 12 each 2.0 ⁇ 1, and 30 ⁇ ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 29 cycles (denaturation at 94 °C for 45 s, annealing at 5 C for 45 s, extension at 72 °C for 45 s), extension at 72 °C for 10 min.
  • M is the DNA Ladder Marker (DL2000, TakaRa), and 1-5 is the drought-tolerant transgenic Arabidopsis thaliana T1 plant (in order: T1D1, T1D2, T1D3, T1D4, T1D5), 6- 10 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant, and 11-14 is a non-transgenic Arabidopsis control.
  • the size of the electrophoresis band of the PCR product shown in the figure is the same as the size of J )H ⁇ (about 380 bp).

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Abstract

L'invention concerne une protéine déhydrine ThDH4 issue de Thellungiella halophila, un gène codant pour celle-ci, et une utilisation du gène dans la culture d'une plante transgénique présentant une tolérance accrue à la sécheresse.
PCT/CN2013/001172 2013-09-27 2013-09-27 Protéine déhydrine dh4 issue de thellungiella halophila, gène codant pour celle-ci, et utilisation du gène Ceased WO2015042747A1 (fr)

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CN201380078595.6A CN105452277B (zh) 2013-09-27 2013-09-27 一种小盐芥脱水素蛋白dh4及其编码基因与应用
PCT/CN2013/001172 WO2015042747A1 (fr) 2013-09-27 2013-09-27 Protéine déhydrine dh4 issue de thellungiella halophila, gène codant pour celle-ci, et utilisation du gène

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PCT/CN2013/001172 WO2015042747A1 (fr) 2013-09-27 2013-09-27 Protéine déhydrine dh4 issue de thellungiella halophila, gène codant pour celle-ci, et utilisation du gène

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007005980A2 (fr) * 2005-07-06 2007-01-11 Cornell Research Foundation Genes de deshydrine et promoteurs propres au cafe
CN101955521A (zh) * 2010-09-21 2011-01-26 中国农业大学 植物耐逆性相关蛋白及其编码基因与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007005980A2 (fr) * 2005-07-06 2007-01-11 Cornell Research Foundation Genes de deshydrine et promoteurs propres au cafe
CN101955521A (zh) * 2010-09-21 2011-01-26 中国农业大学 植物耐逆性相关蛋白及其编码基因与应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HUANG, FAPING ET AL.: "Cloning and sequence analysis of a new dehydrin gene (WZY2) from wheat.", JOURNAL OF NORTHWEST A&F UNIVERSITY ( NAT. SCI. ED., vol. 37, no. 2, 28 February 2009 (2009-02-28), pages 93 - 99 *
RAHMAN, L.N. ET AL.: "Interactions of intrinsically Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes - synergistic effects of lipid composition and temperature on secondary structure.", BIOCHEM. CELL BIOL., vol. 88, no. 5, 24 September 2010 (2010-09-24), pages 791 - 807 *
RAHMAN, L.N. ET AL.: "Interactions of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes at cold and ambient temperatures - Surface morphology and single-molecule force measurements show phase separation, and reveal tertiary and quaternary associations.", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1828, no. 3, 3 December 2012 (2012-12-03), pages 967 - 980 *

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