[go: up one dir, main page]

WO2015042745A1 - Protéine de type déhydrine dh2 de thellungiella halophila, gène codant pour celle-ci et application associée - Google Patents

Protéine de type déhydrine dh2 de thellungiella halophila, gène codant pour celle-ci et application associée Download PDF

Info

Publication number
WO2015042745A1
WO2015042745A1 PCT/CN2013/001170 CN2013001170W WO2015042745A1 WO 2015042745 A1 WO2015042745 A1 WO 2015042745A1 CN 2013001170 W CN2013001170 W CN 2013001170W WO 2015042745 A1 WO2015042745 A1 WO 2015042745A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
gene
plant
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/CN2013/001170
Other languages
English (en)
Chinese (zh)
Inventor
崔洪志
陈文华
孙超
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 CN201380078633.8A priority Critical patent/CN105452282A/zh
Priority to PCT/CN2013/001170 priority patent/WO2015042745A1/fr
Publication of WO2015042745A1 publication Critical patent/WO2015042745A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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 Application in cultivating 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) (this article is named as the coding gene of DH2, and its DNA 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 Dm of a small salt mustard (this article is named
  • ThDH2 the sequence of which is 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)H2-2300 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)H2-2300) of T DH2 (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector C35S-7)H2-2300) of T DH2.
  • Figure 3 shows the drought tolerance simulation results of 7) H2 T1 transgenic Arabidopsis plants (in the figure, T1B3) 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)
  • 1-7 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant (in order: T1B1, T1B2, T1B3, T1B4, T1B5, T1B6, T1B7)
  • 8-11 is not drought tolerant
  • 12-15 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 darkness, stopped watering, and treated for 10 days. After the treatment, the leaves of the top two groups of the seedlings were cut in time. After the nitrogen was rapidly frozen, it was stored in a -70 °C refrigerator.
  • Invitrogen Extracts total RNA from leaves of small salt mustard.
  • 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
  • the total RNA was detected by electrophoresis, and the brightness of the 28S band was about twice that of the 18S band, indicating good RNA integrity.
  • Purification of polyA+ from total RNA using Qiagen's Oligotex mRNA Purification Kit isolates mRNA.
  • 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 a 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 The vector 1 ⁇ 1, ⁇ 4 DNA ligase 1 ⁇ was ligated overnight at 4 °C. Then 10 ⁇ L of the reaction product was added and added to 100 competent E.
  • 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).
  • EST (unigene) 0 has 22 contigs analyzed and 101 single sequences. It was found by BlastN that 53 unigenes have homologous sequences in GenBank, 21 ESTs are unknown or hypothetical proteins, and another 27 have not obtained homologous matches, presumably in the untranslated region at the 3' or 5' end. Short sequence.
  • Example 2 Cloning of a small salt mustard dehydrin protein encoding gene ThDH2
  • SEQ ID NO: 3 is the coding gene 73 ⁇ 4DH2
  • YLS-93GSP1 SEQ ID NO: 4:
  • TTCAGGGTTTCCATGGTGAC YLS-93GSP2 SEQ ID NO: 5:
  • the kit comes with universal primers:
  • AAP SEQ ID NO: 7: GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIGIG
  • 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-93GSP1 (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 step was followed by a Poly C tail, and the first round of PCR amplification was carried out using the tailed product as a template.
  • the primers used were SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (the kit is self-contained, and I is hypoxanthine). Modified a, c, g or t), the specific steps are as follows:
  • PCR reaction system 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ Reverse transcription of the above mRNA, 1.0 ⁇ Ex Taq (purchased from TAKARA), primer of 10 ⁇ 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 58 °C for 45 s, extension at 72 °C for 1 min), 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 universal primer SEQ ID NO: 8.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 l Ex Taq, 10 ⁇ primers SEQ ID NO: 5 and SEQ ID NO: 8 2.0 ⁇ l, and 35 ⁇ 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 58 °C for 45 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • a strip of approximately 460 bp in the second PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into the pGEM-T Easy Vector vector, then transformed into JM109 (specifically the same as above), randomly picked 10 White colonies were inoculated separately in LB liquid medium containing 50 g/mL ampicillin, cultured at 37 ° C overnight, and then glycerin was added to a final concentration of glycerol of 20% by volume, 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), and four positive clones (YL16-1, YL16-3, YL16-5 and YL16-6), sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. to obtain a 5' end sequence of the cDNA of the gene.
  • the obtained 5' RACE product clone YL16-3 was sequenced to obtain the sequence of SEQ ID NO: 9:
  • SEQ ID NO: 10 is the full length sequence of 73 ⁇ 4 DH2.
  • a pair of primers were designed according to the sequence of SEQ ID NO: 10 as follows:
  • ThDH2F SEQ ID NO: 1 1:
  • ThDH2R SEQ ID NO: 12:
  • ThDH2 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 reverse-transcribed, and then the PfuUltra II Fusion HS DNA Polymerase of stratagene was used to obtain the small amount obtained above.
  • the salt mustard cDNA was used as a template for PCR reaction. 50 ⁇ 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 ⁇ l, add 2 volumes of absolute ethanol, -20 ° C placed After 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 P 1.0 ⁇ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes.
  • the approximately 600 bp DNA fragment will be obtained Recycling (Omega Recycling Kit), ligated to pGEM T-easy vector (to obtain ThDH2-pGEM plasmid), then transformed into JM109, and randomly picked 6 white colonies and inoculated separately into LB liquid medium containing 50 g/mL ampicillin. Medium culture, culture overnight at 37 ° C, add glycerol to a final concentration of glycerol 20% by volume, 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 are the same as above), and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, sequence.
  • Is SEQ ID NO: 2 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 Pnos promoter was used to replace the CaMV35S promoter containing the double-enhancer 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 T DH2 gene, and the ThDH2 gene was between the 35 S promoter and the Tnos terminator.
  • Pnos were amplified using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using primers SEQ ID NO: 14 and SEQ ID NO: 15, using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR 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, 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 ⁇ l, and 31 ⁇ 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 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:
  • ThDH2 using the SEQ ID NO: 20 and SEQ ID NO: 21 (template is the positive 73 ⁇ 4 DH2-pGEM plasmid obtained in Example 2) using strafgene's PfuUltra II Fusion HS DNA Polymerase.
  • PCR reaction system 5 ⁇ lOxPfuUltra II reaction Buffer, 0.5 ⁇ l 25 mM dNTP, 2.0 ⁇ ThDH2-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 ⁇ 4DH2-2300.
  • 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 was 0.4, and a seed bacterial liquid was formed.
  • Transformation of Agrobacterium The competent cells were thawed on ice, and 1 ⁇ M of the positive 35S-7)H2-2300 plasmid obtained in Example 3 was added to 40 ⁇ of competent cells, and the mixture was mixed and ice bathed for about 10 min. Transfer the mixture of competent cells and 35S-ThDH2-2300 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.
  • Example 5 Agrobacterium-mediated transformation was used to obtain transgenic Arabidopsis thaliana
  • 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 crucible with a diameter of 7.5 cm containing peat soil and vermiculite (3:1 by volume), 6 plants per pot, placed at 23 ° C, 16 hours light / 8 hours dark Growing in the incubator.
  • 1/2MS medium (9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM H 4 N0 3 , 0.75 mM MgS0 4 , 1.5 mM CaCl 2 , 50 ⁇ , 100 ⁇ 3 ⁇ 3 , 100 MMnSO 4 ) was added to each mash before transplanting. 30 MZnSO 4 , 1 ⁇ Na 2 Mo0 4 , 0.1 MCoCl 2 , 100 ⁇ 2 ⁇ , 100 MFeSO 4 ) 40 ml. After transplanting, the soil moisture should be replenished in time. The nutrient solution is properly watered during the growth period. Every 3-4 weeks (or longer) as needed.
  • 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 mMMgS0 4 , 0.75 mM CaCl 2 , 25 ⁇ , 50 ⁇ 3 ⁇ 3 , 50 MMnSO 4 , 15 MZnS0 4 , 0.5 ⁇ 2 ⁇ 0 4 , 0.05 MCoCl 2 , 50 ⁇ Na 2 EDTA, 50 MFeSO4) Add a 0.8% (mass to volume) agar powder to the aqueous solution of the element, heat it in a microwave oven until the agar is completely dissolved, and cool it to about 50 °C.
  • 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), identified by PCR as Positive plants were numbered (T1B1-T1B12) and stored.
  • Example 6 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis thaliana T1 plants overexpressing ThDH2 The sterilized vermiculite was soaked with 1/2 MS medium.
  • T1B1-T1B6 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, and there were 24 strains of T1B1, T1B2, T1B3, T1B4, T1B5 and T1B6.
  • T1B4, T1B5, T1B6, T1B7 (seven strains) and drought-tolerant transgenic Arabidopsis thaliana T1 plants were each treated with 0.05 g of leaves per day for 10 days, and total RNA was extracted using a plant RNA extraction kit (Invitrogen). Absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate individual RNA concentrations. Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (2 total RNA as a template and reverse transcription primer as SEQ ID NO: 13).
  • the relative expression of DH2 protein was detected by amplifying T DH2 by SEQ ID NO: ll and SEQ ID NO: 12.
  • the PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase.
  • 50 ⁇ PCR 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 ⁇ l, and 30 ⁇ of 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)
  • 1-7 is the drought-tolerant transgenic Arabidopsis thaliana T1 plant (in order: T1B1, T1B2, T1B3, T1B4, T1B5, T1B6, T1B7)
  • 8-11 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant
  • 12-15 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 the 73 ⁇ 4DH2 (about 600 bp).

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

L'invention concerne une protéine de type déhydrine de Thellungiella halophila ThDH2, un gène codant pour celle-ci et une application associée dans la culture d'une plante transgénique présentent une tolérance accrue à la sécheresse.
PCT/CN2013/001170 2013-09-27 2013-09-27 Protéine de type déhydrine dh2 de thellungiella halophila, gène codant pour celle-ci et application associée Ceased WO2015042745A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201380078633.8A CN105452282A (zh) 2013-09-27 2013-09-27 一种小盐芥脱水素蛋白dh2及其编码基因与应用
PCT/CN2013/001170 WO2015042745A1 (fr) 2013-09-27 2013-09-27 Protéine de type déhydrine dh2 de thellungiella halophila, gène codant pour celle-ci et application associée

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2013/001170 WO2015042745A1 (fr) 2013-09-27 2013-09-27 Protéine de type déhydrine dh2 de thellungiella halophila, gène codant pour celle-ci et application associée

Publications (1)

Publication Number Publication Date
WO2015042745A1 true WO2015042745A1 (fr) 2015-04-02

Family

ID=52741710

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/001170 Ceased WO2015042745A1 (fr) 2013-09-27 2013-09-27 Protéine de type déhydrine dh2 de thellungiella halophila, gène codant pour celle-ci et application associée

Country Status (2)

Country Link
CN (1) CN105452282A (fr)
WO (1) WO2015042745A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN102080088A (zh) * 2009-11-27 2011-06-01 创世纪转基因技术有限公司 一种棉花脱水素类似基因及其应用
CN102492030A (zh) * 2011-12-06 2012-06-13 吉林大学 包含脱水素功能域的基因及其抗旱抗碱基因工程应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1544631A (zh) * 2002-11-26 2004-11-10 林忠平 脱水素基因BcDh2及其启动子在培育耐旱植物中的应用
CN1417337A (zh) * 2002-11-26 2003-05-14 林忠平 脱水素基因BcDh2及其启动子在培育耐旱植物中的应用
US20080196126A1 (en) * 2004-06-21 2008-08-14 Eviatar Nevo Transgenic Plants Containing a Dehydrin Gene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102080088A (zh) * 2009-11-27 2011-06-01 创世纪转基因技术有限公司 一种棉花脱水素类似基因及其应用
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
CN102492030A (zh) * 2011-12-06 2012-06-13 吉林大学 包含脱水素功能域的基因及其抗旱抗碱基因工程应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAHMAN, L.N. ET AL.: "Interactions of intrinsically disordered Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes-synergistic effects of lipid composition and temperature on secondary structure", BIOCHEMISTRY AND CELL BIOLOGY, vol. 88, no. 5, 24 September 2010 (2010-09-24), pages 791 - 807 *

Also Published As

Publication number Publication date
CN105452282A (zh) 2016-03-30

Similar Documents

Publication Publication Date Title
WO2014172852A1 (fr) Facteur de transcription myb myb1-1 du coton et son gène codant, et son utilisation
WO2015042742A1 (fr) Protéine de type calcineurine b cbl-3 du coton, gène codant pour celle-ci et utilisation associée
WO2014172826A1 (fr) Pyrophosphatase de tonoplaste vp1 issue de thellungiella halophila, ainsi que gène codant et application associés
WO2015042749A1 (fr) Protéine déhydrine dh5, provenant de thellungiella halophila, gène codant celle-ci et application correspondante
WO2015042740A1 (fr) Protéine de type calcineurine b cbl-4 de thellungiella halophila, gène codant pour celle-ci et application associée
WO2014169472A1 (fr) Protéine bzip-3 de coton de type glissière à leucine, gènes codant pour celle-ci, et application
WO2014172847A1 (fr) Facteur de transcription myb1-2 de type myb de thellungiella salsuginea et gene codant, et son utilisation
WO2014107863A1 (fr) Protéine à fermeture éclair de leucine homéotique du coton hdbzip-1 et gène codant et utilisation de celle-ci
WO2015042738A1 (fr) Protéine glissière à leucine homéotique hdbzip-3 issue de thellungiella halophila, gène codant pour cette protéine, et utilisation du gène
CN105008390B (zh) 一种棉花同源异型‑亮氨酸拉链蛋白HDbZIP‑2及其编码基因与应用
WO2015042745A1 (fr) Protéine de type déhydrine dh2 de thellungiella halophila, gène codant pour celle-ci et application associée
CN105073774B (zh) 一种棉花亮氨酸拉链蛋白bZIP-2及其编码基因与应用
CN105008389B (zh) 一种小盐芥钼辅酶因子硫化酶mcsu-2及其编码基因与应用
WO2014082280A1 (fr) Protéine en doigt de zinc (czf4) de coton, gène la codant et son application
WO2014101153A1 (fr) Isopentényl transférase ipt2 de coton, gène codant pour celle-ci et application de celle-ci
WO2015042747A1 (fr) Protéine déhydrine dh4 issue de thellungiella halophila, gène codant pour celle-ci, et utilisation du gène
WO2015042746A1 (fr) Protéine de type déhydrine dh3 de thellungiella halophila, gène codant pour celle-ci et application associée
WO2015042748A1 (fr) Protéine de type déhydrine dh7 du coton, gène codant pour celle-ci et utilisation associée
WO2015042750A1 (fr) Protéine déhydrine dh6,issue de la thellungiella halophila, gène codant ladite protéine et application correspondante
WO2014172842A1 (fr) Cystéine protéase cysp1-2 du coton et son gène codant, et son utilisation
WO2014082285A1 (fr) Protéine à doigt de zinc du coton (czf5) et son gène codant et son utilisation
WO2014172840A1 (fr) Cystéine protéase cysp1-1 du coton et son gène codant, et son utilisation
WO2015058322A1 (fr) Cofacteur à molybdène sulfurylase mcsu de bruguiera gymnorrhiza, gène codant pour celle-ci et utilisation
WO2015042743A1 (fr) Protéine de type calcineurine b cbl-8 de thellungiella halophila, gène codant pour celle-ci et application associée
WO2015058323A1 (fr) Bétaïne aldéhyde déshydrogénase (badh) de bruguiear gymnorrhiza, gène codant pour celle-ci, et application du gène codeur

Legal Events

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

Ref document number: 201380078633.8

Country of ref document: CN

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

Ref document number: 13894526

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: 13894526

Country of ref document: EP

Kind code of ref document: A1