WO2015042747A1 - Thellungiella halophila dehydrin protein dh4, coding gene of same, and application thereof - Google Patents

Thellungiella halophila dehydrin protein dh4, coding gene of same, and application thereof 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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PCT/CN2013/001172
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French (fr)
Chinese (zh)
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陈文华
孙超
崔洪志
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创世纪转基因技术有限公司
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Priority to PCT/CN2013/001172 priority Critical patent/WO2015042747A1/en
Priority to CN201380078595.6A priority patent/CN105452277B/en
Publication of WO2015042747A1 publication Critical patent/WO2015042747A1/en

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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

Disclosed are a Thellungiella halophila-sourced dehydrin protein ThDH4, a coding gene of same, and an application thereof in cultivating a transgenic plant of increased drought tolerance.

Description

种小盐芥脱水素蛋白 H^及其编码基因与应用 技术领域 本发明涉及植物蛋白及其编码基因与应用， 特别是涉及一个来源于小盐芥的脱 水素蛋白 /¾ 及其编码基因， 以及其在培育耐旱性提高的转基因植物中的应用。 背景技术  FIELD OF THE INVENTION 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
温度、 盐渍和干旱等逆境胁迫会对高等植物的生长发育造成严重危害， 导致作物产 量降低， 品质下降， 严重威胁农业生产和自然环境。 其中干旱对作物产量的影响， 在诸 多自然逆境中占首位， 其危害相当于其它灾害之和， 是许多地区农业发展的瓶颈。 据统 计， 世界干旱、 半干旱地区占陆地面积的 34%; 我国干旱、 半干旱地区约占国土面积的 52%, 年受旱面积达 200— 270万公顷， 全国灌溉区每年缺水约 30亿立方米， 因缺水而 少收粮食 350— 400亿公斤； 特别是我国主要产粮区如华北、 东北和西北， 是我国缺水最 严重的地区， 春旱频繁达到十年九遇。  Stresses such as temperature, salt and drought can cause serious damage to the growth and development of higher plants, resulting in reduced crop yields, degraded quality, and serious threats to agricultural production and the natural environment. Among them, the impact of drought on crop yields ranks first in many natural adversities, and its harm is equivalent to the sum of other disasters, which is the bottleneck of agricultural development in many regions. According to statistics, the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid areas account for about 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares. Cubic meters, due to lack of water, less than 350-40 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the most severe areas in China, and spring droughts frequently reach 10 years.
植物耐旱性大多属于多基因控制的数量性状， 利用常规育种方法改良作物的抗旱性 受到周期长、 优异种质资源缺乏的限制。 近年来的转录组学、 蛋白组学和基因表达调控 的研究初步揭示了植物干旱胁迫的作用分子机理。 目前， 利用干旱胁迫相关基因提高植 物的抗旱能力， 已经成为植物抗逆分子生物学的研究热点和植物抗逆基因工程重要的研 究方向。  Most of the drought tolerance of plants belongs to the quantitative traits controlled by multiple genes. The use of conventional breeding methods to improve the drought resistance of crops is limited by the long cycle and lack of excellent germplasm resources. Recent studies on transcriptomics, proteomics and gene expression regulation have revealed the molecular mechanism of plant drought stress. At present, the use of drought stress-related genes to improve the drought resistance of plants has become a research hotspot of plant resistance to molecular biology and an important research direction of plant stress resistance genetic engineering.
植物受到逆境胁迫时会产生相应的应答反应， 以降低或消除逆境胁迫给植物带来的 危害。 植物的这种应答反应是一个涉及多基因、 多信号途径及多基因产物的复杂过程。 但就目前的研究状况而言， 由于其机制十分复杂， 许多植物对逆境的生物化学和生理学 响应机制仍有待深入研究。 在抗逆应答基因的功能及表达调控方面的研究将对植物抗逆 相关的信号传递途径之间的联系以及整个信号传递网络***的研究提供重要的基础。 发明内容 本发明人利用 SSH (抑制差减杂交） 与 RACE ( cDNA末端快速扩增） 相结合的方 法克隆了小盐芥的一个脱水素蛋白 （本文命名为 DH4 的编码基因， 并测定了其 DNA 序列。 并且发现将其导入植物超量表达后， 可明显改善转基因植株的耐旱性， 而且这些 性状可稳定遗传。 When plants are stressed by stress, they will respond accordingly to reduce or eliminate the damage caused by stress. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress remain to be further studied. Studies on the function and expression regulation of stress-responsive genes will provide an important basis for the link between plant stress-resistance-related signaling pathways and the study of the entire signaling network system. SUMMARY OF THE INVENTION 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.
本发明第一方面提供小盐芥的一个脱水素蛋白 DH4 的编码基因 （本文命名为 ThDH4) ， 其序列为 SEQ ID NO: 2。  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.
本发明第二方面提供一种重组表达载体， 其含有本发明第一方面所述的基因并且所 述基因的核苷酸序列与所述表达载体的表达控制序列可操作地连接； 优选地， 所述载体 为附图 2所示的 35S-7 )H¥-2300载体。  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.
本发明第三方面提供一种重组细胞， 其含有本发明第一方面所述的基因或者本发明 第二方面所述的重组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。  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.
本发明第四方面提供一种改善植物耐旱性的方法， 包括： 将本发明第一方面所述的 基因或者本发明第二方面所述的重组表达载体导入植物或植物组织并使所述基因表达； 优选地， 所述植物是拟南芥。  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; Preferably, the plant is Arabidopsis thaliana.
本发明第五方面提供一种制备转基因植物的方法， 包括： 在有效产生植物的条件下 培养含有本发明第一方面所述的基因或者本发明第二方面所述的重组表达载体的植物或 植物组织； 优选地， 所述植物是拟南芥。  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; Preferably, the plant is Arabidopsis thaliana.
本发明第六方面提供本发明第一方面所述的基因、 本发明第二方面所述的重组表达 载体或者本发明第三方面所述的重组细胞用于改善植物耐旱性以及用于植物育种的用途; 优选地， 所述植物是拟南芥。  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; Preferably, the plant is Arabidopsis thaliana.
本发明第七方面提供本发明第一方面所述的基因编码的蛋白质， 其氨基酸序列如 SEQ ID NO: 1所示。 附图说明 图 1是 T DH4的植物表达载体 C35S-7 )H¥-2300)的构建流程 （图 la-lb)。  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. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector C35S-7)H ¥-2300) of T DH4 (Fig. la-lb).
图 2是 T DH4的植物表达载体 C35S-7 )H¥-2300)的质粒图。  Figure 2 is a plasmid map of the plant expression vector C35S-7)H¥-2300) of T DH4.
图 3是 7 )H¥ T1代转基因拟南芥植株 （图中， T1D2) 和作为对照的非转基因拟南 芥植株 （图中， CK ) 的耐旱模拟实验结果。 （图 3a为正常生长 20天的拟南芥植株； 图 3b为正常生长 20天后干旱处理 14天的拟南芥植株）。  Figure 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).
图 4是转基因 T1代拟南芥植株和非转基因对照植株在转录水平上的蛋白表达验 证结果。 M为 DNA Ladder Marker ( DL2000, TakaRa) , 1-5为耐旱转基因拟南芥 Tl 代植株 （依次为： T1D1、 T1D2、 T1D3、 T1D4、 T1D5 )， 6-10为不耐旱转基因拟南芥 Tl代植株， 11-14为非转基因拟南芥对照。 具体实施方式 下面结合非限制性实施例对本发明进行进一步说明。所述实施例仅出于示例性目的， 并非意在限制本发明的范围。 Figure 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.
下面实施例中提到的限制性内切酶均购自 New England Biolabs公司。 实施例 1、 干旱胁迫下小盐芥 SSH文库构建：  The restriction enzymes mentioned in the examples below were all purchased from New England Biolabs. Example 1. Construction of SSH library of small salt mustard under drought stress:
具体方法为：  The specific method is:
利用 Clontech公司的 PCR-select™ cDNA Subtraction Kit所示的方法通过抑制差减杂 交方法构建差减文库。在实验中以生长过程中干旱处理的小盐芥幼苗的叶片的 mRNA作 为样本 （tester), 以未处理的小盐芥幼苗的叶片的 mRNA作为对照 （driver)。 具体步骤 如下：  A subtractive library was constructed by the method of inhibition subtractive hybridization using the method shown by Clontech's PCR-selectTM cDNA Subtraction Kit. In the experiment, 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:
( 1 ) 供试材料：  (1) Test materials:
小盐芥 （ T ellungiella halophila, 购自中国内蒙古巴彦淖尔市乌兰布和沙漠绿色 植物园盐生植物繁育中心） ， 播种到灭过菌的蛭石上， 在 25 °C、 光周期 16小时光照 /8小时黑暗（光强 2000— 3000 Lx)条件下培养，每周浇 1/2MS培养基（ 9.39 mM KN03， 0.625 mM KH2P04， 10.3 mM NH4N03 , 0.75 mM MgS04， 1.5 mM CaCl2， 50 μΜ KI， 100 μΜ Η3ΒΟ3， 100 M MnSO4， 30 μΜ ZnS04, 1 μΜ Να2Μο04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 M FeSO4) —次。 当苗株培养 1个月左右时用于实验。 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 μΜ KI, 100 μΜ Η 3 ΒΟ 3 , 100 M MnSO 4 , 30 μΜ ZnS0 4 , 1 μΜ Να 2 Μο0 4 , 0.1 μΜ CoCl 2 , 100 μΜ Na 2 EDTA, 100 M FeSO 4 ). When the seedlings were cultured for about 1 month, they were used for experiments.
( 2 ) 材料处理：  (2) Material handling:
将供试幼苗分为 2组， 每组 4盆， 每盆 1株。 第一组为对照组， 在 25 °C、 光周 期 16小时光照 /8小时黑暗条件下培养， 正常浇灌。 第二组为干旱处理组， 25 °C、 光 周期 16小时光照 /8小时黑暗条件下培养， 停止浇灌， 处理 10天， 处理完毕后及时剪 取两组幼苗顶端的叶片， 用液氮迅速冷冻后， 于 -70 °C冰箱中保存。  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.
( 3 ) 总 RNA提取：  (3) Total RNA extraction:
分别取对照组和干旱处理组的小盐芥叶片 0. 1g， 用植物 RNA提取试剂盒 （购自 Invitrogen) 提取小盐芥叶片的总 RNA。 用 HITACHI公司的紫外分光光度计 U-2001 测定总 RNA在 260 nm和 280 nm的吸光度值， OD260/OD280比值为 1.8-2.0， 表明总 RNA纯度较高； 用 1.0%的琼脂糖凝胶电泳检测总 RNA的完整性， 28S条带的亮度 约为 18S条带的 2倍，表明 RNA的完整性良好。使用 Qiagen 公司的 Oligotex mRNA 纯化试剂盒（purification of poly A+ RNA from total RNA, 从总 RNA中纯化 polyA+ RNA)分离 mRNA。 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).
( 4 ) 抑制差减杂交：  (4) Suppression of subtractive hybridization:
按 Clontech公司的 PCR-selectTM cDNA Subtraction Kit试剂盒所示的方法进行抑制差 减杂交。先将 Driver mRNA和 Tester mRNA分别反转录，得到双链 cDNA,再以 2 Tester cDNA禾 P 2 g Driver cDNA作为起始材料进行差减杂交。 在 37°C水浴下分别将 Tester cDNA和 Driver cDNA用 Rsa I 酶切 1.5 h， 然后将酶切后的 Tester cDNA分成两等份， 连接上不同的接头， 而 Driver cDNA不连接头。 两种连有不同接头的 Tester cDNA分别 与过量的 Driver cDNA混合， 进行第一次正向差减杂交。 将两种第一次正向差减杂交的 产物混合， 再与新变性的 Driver cDNA进行第二次正向差减杂交， 然后通过两次抑制性 PCR扩增差异表达的片段， 使其得到富集。 Suppression Subtractive Hybridization performed by PCR-select TM cDNA Clontech's method shown Subtraction Kit kit. The Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and then subtracted hybridization using 2 Tester cDNA and P 2 g Driver cDNA as starting materials. 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 two products of the first forward subtractive hybridization 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 a rich set.
为了增加获得表达序列标签 （Expressed sequence tag, EST) (unigene)的有效性， 避 免基因无酶切位点及所获得序列在非翻译区， 本实验同时用内切酶 Haelll按上述步骤对 Tester cDNA和 Driver cDNA进行酶切并先后进行两次正向差减杂交和两次抑制性 PCR 扩增， 最后合并两组正向差减杂交 cDNA片段的第二次抑制性 PCR产物。  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.
( 5 ) cDNA差减文库的构建与初步筛选、 克隆、 鉴定  (5) Construction and preliminary screening, cloning and identification of cDNA subtraction library
依照 pGEM-T Easy试剂盒（购自 Promega)的说明，将上述合并的正向差减杂交 cDNA 片段的第二次 PCR产物 （使用 QIAquick PCR Purification Kit纯化， 购自 Qiagen) 与 pGEM-T Easy载体连接， 其具体步骤如下： 在 200 μΐ PCR管中依次加入下列成分： 纯化 的正向差减杂交 cDNA片段的第二次 PCR产物 3 μ1、 2χΤ4连接酶缓冲液 5 μ1、 pGEM-T Easy载体 1 μ1、 T4 DNA连接酶 1 μ1， 于 4°C连接过夜。然后取 10 连接反应产物， 加 入到 100 μL感受态大肠杆菌 JM109 (购自 TAKARA)中，冰浴 30 min、 42°C热休克 60 s、 冰浴 2 min,另加 250 μL LB液体培养基（含有 1%胰蛋白胨（Tryptone,购自 OXOID)、 0.5% 酵母提取物 （Yeast Extract, 购自 OXOID ) 禾 P 1% NaCl (购自国药）） 置于 37°C 摇床中， 以 225 r/min振荡培养 30 min, 然后从中取 200 μ 菌液涂布于含 50 g/ml氨苄 青霉素、 40 g/mL X-gal、 24 g/mL IPTG (X-gal ( 5-溴 -4氯 -3-吲哚 - β -D-半乳糖苷) 和 IPTG (异丙基 - β -D-硫代吡喃半乳糖苷） 购自 TAKARA) 的 LB固体培养板上， 37°C培 育 18小时。 计数培养板中直径 > 1 mm的清晰白色及蓝色菌落数， 随机挑取 198个白色 菌落 (编号： YLS-001至 YLS-198)。 将所挑取的白色菌落分别接种于 96 孔细胞培养板 (CORNING)中含有 50 g/mL氨苄青霉素的 LB液体培养基， 37°C培养过夜后加甘油至甘 油终浓度 20% (体积比）， 于 -80°C保存备用。 对所培养的菌落克隆以巢式 PCR (引物 Primer 1和 Primer 2R, 来自 Clontech公司的 PCR-select™ cDNA Subtraction Kit试剂盒） 进行菌液 PCR扩增验证，得到 166个阳性克隆，然后将所有阳性克隆送英潍捷基 (上海） 贸易有限公司测序。 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) For the ligation, the specific steps are as follows: The following components are sequentially added to the 200 μΐ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 μ1, 2χΤ4 ligase buffer 5 μ1, pGEM-T Easy vector 1 Μ1, T4 DNA ligase 1 μl, and ligated overnight at 4 °C. Then, 10 reaction products were added and added to 100 μL 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 μL 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 μ 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). - 吲哚-β-D-galactoside) and IPTG (isopropyl-β-D-thiogalactopyranoside) were purchased from LB solid plates on TAKARA) and incubated at 37 ° C for 18 hours. Count the number of clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 198 white colonies (number: YLS-001 to YLS-198). Inoculate the picked white colonies in a 96-well cell culture plate (CORNING) LB liquid medium containing 50 g/mL ampicillin, cultured at 37 ° C overnight, then added with glycerol to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C for later use. 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.
(6) 差异克隆的 cDNA测序分析：  (6) cDNA sequencing analysis of differential clones:
将 DNA 测序结果去除载体和不明确序列及冗余的 cDNA 后， 共得到 123 个 EST(unigene)0 经分析有 22个重叠群， 有 101个单一的序列。 经 BlastN发现其中 53条 unigene在 GenBank 中有同源序列， 21条 EST功能未知或者为假定蛋白， 另有 27条未 获得同源匹配， 推测可能是处于 3'或 5'末端非翻译区的较短序列。 实施例 2 盐芥脱水素蛋白编码基因 ThDH4的克隆 After removing the carrier and DNA sequencing uncertainty of the cDNA sequence and the redundancy, to give a total of 123 EST (unigene) 0 analyzed contigs 22, 101 has a single sequence. 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 the salt devonian protein encoding gene ThDH4
克隆子 YLS-113去掉冗余 DNA后， 序列为 SEQIDNO: 3， 序列分析表明该序列的 编码的蛋白质属于脱水素蛋白， 本文将克隆子 YLS-113 对应的全长编码基因命名为 ThDH4, 其对应的蛋白命名为 DH¥。  After the cloned YLS-113 was detached from the redundant DNA, the sequence was SEQ ID NO: 3. Sequence analysis indicated that the encoded protein of the sequence belonged to the dehydrin protein. In this paper, the full-length coding gene corresponding to the clone YLS-113 was named ThDH4, corresponding to The protein is named DH¥.
SEQIDNO: 3 SEQIDNO: 3
1 GCTCGGAGGA CGAGGGACTA GGTGGAAGGA CGAAGAAAAA GGGAATAACG GAGAAGATAA1 GCTCGGAGGA CGAGGGACTA GGTGGAAGGA CGAAGAAAAA GGGAATAACG GAGAAGATAA
61 AGGAGAAGCT GCCATGTGGT CATCATGGGT CCCACCAGAC TTCTTCAGCG ATTGGCAACA 121 CCACGGCTTA TGATACTACT GGCACCGGCG CCGTTCACGA CGAGAAGAAA GGCATTGTGG61 AGGAGAAGCT GCCATGTGGT CATCATGGGT CCCACCAGAC TTCTTCAGCG ATTGGCAACA 121 CCACGGCTTA TGATACTACT GGCACCGGCG CCGTTCACGA CGAGAAGAAA GGCATTGTGG
181 AGAAGATCAT GGACAAGCTT CCCGGTGGCC ATTATTAG 181 AGAAGATCAT GGACAAGCTT CCCGGTGGCC ATTATTAG
DH4全长编码基因的克隆 Cloning of the full-length coding gene of DH4
根据已经获得的 SEQIDNO: 3序列分析： SEQIDNO: 3 为编码基因 7 )H¥ 的 3' 端序列。 根据已经获得的 SEQIDNO: 3序列， 设计如下三条特异性引物， 作为反转录引 物及 5 'RACE的特异性引物。  According to the sequence analysis of SEQ ID NO: 3 which has been obtained: 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.
YLS-113GSP1: SEQIDNO: 4: YLS-113GSP1: SEQIDNO: 4:
TCGTGAACGGCGCCGGTG TCGTGAACGGCGCCGGTG
YLS-113GSP2: SEQIDNO: 5: YLS-113GSP2: SEQIDNO: 5:
TGTTGCCAATCGCTGAAGAAG YLS-113GSP3: SEQ ID NO:6: GCTCGGAGGACGAGGGACTAG TGTTGCCAATCGCTGAAGAAG YLS-113GSP3: SEQ ID NO:6: GCTCGGAGGACGAGGGACTAG
试剂盒自带通用引物：  The kit comes with universal primers:
AAP: SEQ ID NO: 7：  AAP: SEQ ID NO: 7:
GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP: SEQ ID NO: 8：  GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP: SEQ ID NO: 8:
GGCCACGCGTCGACTAGTAC 实验步骤按试剂盒说明书操作 （5' RACE System for Rapid Amplification of cDNA Ends试剂盒购自 Invitrogen公司）。  The GGCCACGCGTCGACTAGTAC experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
以 YLS-113GSP1(SEQ ID NO: 4)为反转录引物， 以干旱处理组小盐芥叶片提取的 mRNA为模板进行反转录， 获得 cDNA模板， 然后按照上述 5' RACE试剂盒说明书中的 步骤加 PolyC尾，以加尾后的产物为模板进行第一轮 PCR扩增，所用引物为 SEQIDNO: 4与通用引物 SEQ ID NO: 7 (试剂盒自带， I为次黄嘌吟修饰的 a、 c、 g或 t)， 具体步 骤如下：  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:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ mRNA反转录 的 cDNA、 1.0 μΐ Ex Taq (购自 TAKARA)、 10 μΜ的引物 SEQ ID NO: 4和 SEQ ID NO: 7 各 2.0 μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环 （94°C 变性 45s， 62°C退火 45 s， 72°C延伸 45s)， 72°C延伸 10min。  50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΐ Ex Taq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 4 and SEQ ID NO: 7 each of 2.0 μl, and 35 μΐ 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.
所得的 PCR产物用双蒸水稀释 50倍后取 2.0 μΐ作为模板， 用 SEQ ID NO: 5与通 用引物 SEQIDNO: 8进行第二轮 PCR扩增， 具体步骤如下：  The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL 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:
50 μ1 ΡΟ 反应体系： 5 μΐ 10XExBuffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ稀释的第 一轮 PCR产物、 1.0 μΐ Ex Taq、10 μΜ的引物 SEQ ID NO: 5禾 P SEQ ID NO: 8各 2.0 μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环 （94°C变性 45 s， 60°C退火 45 s， 72°C延伸 1 min)， 72°C延伸 10min。回收第二次 PCR产物中约为 290bp 大小的条带 （ Gel Extraction Kit购自 OMEGA), 并将其连接到 pGEM-T Easy Vector载 体， 然后转化到 JM109(具体方法同上）， 随机挑取 10 个白色菌落分别接种于含有 50 g/mL氨苄青霉素的 LB液体培养基中， 37°C培养过夜后加甘油至甘油终浓度 20% (体 积比），-80°C保存备用。用引物 SEQ ID NO: 5与 3'端引物 SEQ ID NO: 6进行菌液 PCR扩 增 （反应体系及反应条件同上） 验证， 得到 3个阳性克隆 （01、 03和 06)， 送英潍捷基 (上海） 贸易有限公司测序， 获得该基因的 cDNA的一段 5'端序列。  50 μl ΡΟ Reaction system: 5 μΐ 10X ExBuffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 μΐ Ex Taq, 10 μΜ primer SEQ ID NO: 5 and P SEQ ID NO: 8 each 2.0 11, and 35 μΐ 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 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.
所得的 5'RACE产物克隆子 06测序获得序列为 SEQ IDNO: 9： 1 GGGGGGGGGG ATGGAGTCTA ATTATCAAAA CCAATCGGGA GCGCAGCAGA GTCACCAACAThe resulting 5' RACE product clone 06 was sequenced to obtain the sequence of SEQ ID NO: 9: 1 GGGGGGGGGG ATGGAGTCTA ATTATCAAAA CCAATCGGGA GCGCAGCAGA GTCACCAACA
61 GCTTGACCAA TACGGAAATC CAGTCCCAGT CGGAACCGGA GTCTATGCAG CTCCGGTCAT61 GCTTGACCAA TACGGAAATC CAGTCCCAGT CGGAACCGGA GTCTATGCAG CTCCGGTCAT
121 GGCTGGTGGC CTGCTTCATC GTTCTGGAAG TAGCTCTAGC TCTAGCTCTA GCTCGGAGGA121 GGCTGGTGGC CTGCTTCATC GTTCTGGAAG TAGCTCTAGC TCTAGCTCTA GCTCGGAGGA
181 CGAGGGACTA GGTGGAAGGA CGAAGAAAAA GGGAATAACG GAGAAGATAA AGGAGAAGCT181 CGAGGGACTA GGTGGAAGGA CGAAGAAAAA GGGAATAACG GAGAAGATAA AGGAGAAGCT
241 GCCATGTGGT CATCATGGGT CCCACCAGAC TTCTTCAGCG ATTGGCAACA 将 5 'RACE获得的序列 SEQ ID NO : 9， 与获得的序列 SEQ ID NO : 3拼接， 获得 SEQ ID NO: 10 : 241 GCCATGTGGT CATCATGGGT CCCACCAGAC TTCTTCAGCG ATTGGCAACA The sequence of SEQ ID NO: 9, obtained by 5 'RACE, was spliced with the obtained sequence SEQ ID NO: 3 to obtain SEQ ID NO: 10:
1 GGGGGGGGGG ATGGAGTCTA ATTATCAAAA CCAATCGGGA GCGCAGCAGA GTCACCAACA 1 GGGGGGGGGG ATGGAGTCTA ATTATCAAAA CCAATCGGGA GCGCAGCAGA GTCACCAACA
61 GCTTGACCAA TACGGAAATC CAGTCCCAGT CGGAACCGGA GTCTATGCAG CTCCGGTCAT61 GCTTGACCAA TACGGAAATC CAGTCCCAGT CGGAACCGGA GTCTATGCAG CTCCGGTCAT
121 GGCTGGTGGC CTGCTTCATC GTTCTGGAAG TAGCTCTAGC TCTAGCTCTA GCTCGGAGGA121 GGCTGGTGGC CTGCTTCATC GTTCTGGAAG TAGCTCTAGC TCTAGCTCTA GCTCGGAGGA
181 CGAGGGACTA GGTGGAAGGA CGAAGAAAAA GGGAATAACG GAGAAGATAA AGGAGAAGCT181 CGAGGGACTA GGTGGAAGGA CGAAGAAAAA GGGAATAACG GAGAAGATAA AGGAGAAGCT
241 GCCATGTGGT CATCATGGGT CCCACCAGAC TTCTTCAGCG ATTGGCAACA CCACGGCTTA241 GCCATGTGGT CATCATGGGT CCCACCAGAC TTCTTCAGCG ATTGGCAACA CCACGGCTTA
301 TGATACTACT GGCACCGGCG CCGTTCACGA CGAGAAGAAA GGCATTGTGG AGAAGATCAT301 TGATACTACT GGCACCGGCG CCGTTCACGA CGAGAAGAAA GGCATTGTGG AGAAGATCAT
361 GGACAAGCTT CCCGGTGGCC ATTATTAG 根据 SEQ ID NO : 10序列分析， SEQ ID NO: 10 为 7¾DH¥的全长序列。 根据 SEQ ID361 GGACAAGCTT CCCGGTGGCC ATTATTAG According to the sequence analysis of SEQ ID NO: 10, SEQ ID NO: 10 is the full length sequence of 73⁄4 DH¥. According to SEQ ID
NO: 10 序列设计一对引物如下： NO: 10 Sequence Design A pair of primers are as follows:
ThDH4F : SEQ ID NO: 1 1：  ThDH4F : SEQ ID NO: 1 1:
ATGGAGTCTAATTATCAAAACC  ATGGAGTCTAATTATCAAAACC
ThDH4R: SEQ ID NO: 12： ThDH4R: SEQ ID NO: 12:
CTAATAATGGCCACCGGGA AP : SEQ ID NO: 13：  CTAATAATGGCCACCGGGA AP : SEQ ID NO: 13:
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 通过 SEQ ID NO: 11和 SEQ ID NO: 12来克隆 ThDH4全长编码序列。  GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT The ThDH4 full-length coding sequence was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
提取干旱处理组小盐芥的 RNA作为模板， 以引物 SEQ ID NO: 13为反转录引物， 反转录获取小盐芥 cDNA，然后采用 stratagene的 PfuUltra II Fusion HS DNA Polymerase, 以上述获得的小盐芥的 cDNA 为模板进行 PCR 反应。 50 μΐ PCR 反应体系： 5 μΐ 10 X PfuUltra II reaction Buffer 0.5 μΐ 25 mM的 dNTP、 2.0 μΐ cDNA 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase、 10 μΜ的引物 SEQ ID NO: 1 1和 SEQ ID NO: 12各 2.0 μ1， 以及 37.5 μΐ的双蒸水。 PCR反应条件： 95 °C预变性 2 min, 35个循环（95 °C变性 25 s， 51 °C退火 25 s， 72°C延伸 30min) ， 72°C延伸 5 min。  The 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. 50 μΐ PCR reaction system: 5 μΐ 10 X PfuUltra II reaction Buffer 0.5 μΐ 25 mM dNTP, 2.0 μΐ cDNA 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase, 10 μΜ primers SEQ ID NO: 1 1 and SEQ ID NO: 12 2.0 μ1, and 37.5 μΐ 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扩增产物加 A尾： PCR产物补水至 400μ1， 先用氯仿抽提一遍去除蛋白， 吸 取上清加入 3Μ 醋酸钠溶液 40μ1， 加入 2倍体积的无水乙醇， -20°C放置 10分钟， 离 心， 去上清， 晾干， 用 21 μΐ双蒸水溶解。 加入 2.5 μΐ Ι Ο Χ Εχ Buffer 0.5 μΐ 5 mM的 dATP和 1.0 μΐ Ex Taq。 反应条件： 70°C反应 30分钟。将得到的约 380bp的 DNA片段 回收（Omega回收试剂盒），连接至 pGEM T-easy载体上（得到 ThDH4-pGEM质粒）， 然后转化 JM109, 随机挑取 8个白色菌落分别接种于含有 50 g/mL氨苄青霉素的 LB 液体培养基中培养， 37°C培养过夜后加甘油至甘油终浓度 20% (体积比） ， -80°C保存 备用。 用引物 SEQ ID NO: 1 1与 SEQ ID NO: 12进行菌液 PCR扩增 （反应体系及反应 条件同上） ， 得到 3个阳性克隆， 送至英潍捷基 （上海） 贸易有限公司测序， 序列为 SEQ ID NO: 2， 其编码的蛋白的氨基酸序列为 SEQ ID NO: 1 PCR amplification product plus A tail: PCR product hydration to 400μ1, first remove the protein with chloroform extraction, add the supernatant to the 3 Μ sodium acetate solution 40μ1, add 2 volumes of absolute ethanol, -20 ° C for 10 minutes, Centrifuge, remove the supernatant, allow to dry, and dissolve in 21 μl of double distilled water. Add 2.5 μΐ Ι Ο Χ Εχ Buffer 0.5 μΐ 5 mM dATP and 1.0 μΐ 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. Is SEQ ID NO: 2, the amino acid sequence of the encoded protein is SEQ ID NO: 1
DH4蛋白的氨基酸序列： SEQ ID NO: 1： Amino acid sequence of DH4 protein: SEQ ID NO: 1:
1 ESNYQNQSG AQQSHQQLDQ YGNPVPVGTG VYAAPVMAGG LLHRSGSSSS SSSSSEDEGL 1 ESNYQNQSG AQQSHQQLDQ YGNPVPVGTG VYAAPVMAGG LLHRSGSSSS SSSSSEDEGL
61 GGRRKKKGIT EKIKEKLPCG HHGSHQTSSA IGNTTAYDTT GTGAVHHEKK GIVEKI DKL 121 PGGHY 61 GGRRKKKGIT EKIKEKLPCG HHGSHQTSSA IGNTTAYDTT GTGAVHHEKK GIVEKI DKL 121 PGGHY
7¾DH¥编码基因的核苷酸序列： SEQ ID NO: 2 Nucleotide sequence of the 73⁄4DH¥ coding gene: SEQ ID NO: 2
1 ATGGAGTCTA ATTATCAAAA CCAATCGGGA GCGCAGCAGA GTCACCAACA GCTTGACCAA 61 TACGGAAATC CAGTCCCAGT CGGAACCGGA GTCTATGCAG CTCCGGTCAT GGCTGGTGGC1 ATGGAGTCTA ATTATCAAAA CCAATCGGGA GCGCAGCAGA GTCACCAACA GCTTGACCAA 61 TACGGAAATC CAGTCCCAGT CGGAACCGGA GTCTATGCAG CTCCGGTCAT GGCTGGTGGC
121 CTGCTTCATC GTTCTGGAAG TAGCTCTAGC TCTAGCTCTA GCTCGGAGGA CGAGGGACTA121 CTGCTTCATC GTTCTGGAAG TAGCTCTAGC TCTAGCTCTA GCTCGGAGGA CGAGGGACTA
181 GGTGGAAGGA GGAAGAAAAA GGGAATAACG GAGAAGATAA AGGAGAAGCT GCCATGTGGT181 GGTGGAAGGA GGAAGAAAAA GGGAATAACG GAGAAGATAA AGGAGAAGCT GCCATGTGGT
241 CATCATGGGT CCCACCAGAC TTCTTCAGCG ATTGGCAACA CCACGGCTTA TGATACTACT241 CATCATGGGT CCCACCAGAC TTCTTCAGCG ATTGGCAACA CCACGGCTTA TGATACTACT
301 GGCACCGGCG CCGTTCACCA CGAGAAGAAA GGCATTGTGG AGAAGATCAT GGACAAGCTT 361 CCCGGTGGCC ATTATTAG 实施例 3 7¾£)//¥基因植物表达载体构建 301 GGCACCGGCG CCGTTCACCA CGAGAAGAAA GGCATTGTGG AGAAGATCAT GGACAAGCTT 361 CCCGGTGGCC ATTATTAG Example 3 73⁄4£)//¥ Gene plant expression vector construction
选择植物双元表达载体 pCAMBIA2300 (购自北京鼎国昌盛生物技术有限责任公 司）作为植物表达载体， 用 Pnos启动子替换 ΝΡΤΠ基因含双增强子的 CaMV35S启动 子， 以降低 ΝΡΤΠ蛋白在植物中的表达。 在 Pnos启动子的上游*** 35S启动子及终 止子 Tnos分别作为 ThDH4基因的启动子和终止子， 7¾DH¥基因在所述 35S启动子和 Tnos终止子之间。  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 ΝΡΤΠ 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.
用引物 SEQ ID NO: 14和 SEQ ID NO: 15以植物表达载体 pBI121 (购自北京华夏 远洋科技有限公司） 为模板扩增 Pnos, 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ1 ΡΟ 反应体系： 10 μΐ 5 xPS Buffer、 3 μΐ 2.5 mM的 dNTP、 1.0 μΐ ρΒΙ121 1.0 μΐ PrimeSTAR HS DNA聚合酶、 10 μΜ的引物 SEQ ID NO: 14和 SEQ ID NO: 15各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环（94°C变性 30 s， 56°C退火 30 s， 72°C延伸 30 s)， 72°C延伸 10 min。 通过 EcoRI、 Bglll酶切后将所得 PCR产物连接到 pCAMBIA2300 ( Promega, T4连接酶盒） 获得 pCAMBIA2300-l。 SEQ ID NO: 14 ： 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 μl ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ ρΒΙ121 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 14 and SEQ ID NO: 15 each 2.0 μl, and 31 μΐ 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. SEQ ID NO: 14:
GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 15 :  GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 15 :
ATCCAGATCTAGATCCGGTGCAGATTATTTG 使用引物 SEQ ID NO: 16和 SEQ ID NO: 17以 pBI121为模板扩增 Tnos， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM的 dNTP、 1.0 μΐ pBI121、 1.0 μΐ PrimeSTAR HS DNA聚合酶、 10 μΜ的引物 SEQ ID NO: 16和 SEQ ID NO: 17各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预 变性 5 min， 33个循环 （94°C变性 30 s， 58°C退火 30 s， 72°C延伸 30 s)， 72 °C延伸 10 min。 通过 Sacl、 EcoRI酶切后将所得 PCR产物连接到 pCAMBIA2300-l ( Promega T4 连接酶盒） 获得 pCAMBIA2300-2。  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 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μl, and 31 μΐ 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.
SEQ ID NO: 16： SEQ ID NO: 16:
AAGG^GCJCGAATTTCCCCGATCGTTCAAA  AAGG^GCJCGAATTTCCCCGATCGTTCAAA
SEQ ID NO: 17：  SEQ ID NO: 17:
TCKGAA rrCCC AGTGAATTCCCGATCT AGT A 使用引物 SEQ ID NO: 18和 SEQ ID NO: 19以 pCAMBIA2300质粒为模板扩增 35S 启动子。采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΙ ΡΟ 反应体系： 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM的 dNTP、 1.0 μΐ稀释 50倍的 pCAMBIA2300质粒、 1.0 μΐ PrimeSTAR HS DNA聚合酶、 10 的引物 8£0 10 ^^0: 18和 8£0 10 ^^0: 19各2.0 ^， 以及 31 μΐ 的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 （94°C变性 30 s， 50°C退火 30 s， 72°C延伸 30 s)， 72°C延伸 10 min。 通过 HindIII、 Pstl酶切后将所得 PCR产物 连接到 （连接方法同上） pCAMBIA2300-2获得 pCAMBIA2300-3 SEQ ID NO: 18：  TCKGAA rrCCC AGTGAATTCCCGATCT AGT A 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 μΙ ΡΟ Reaction system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ diluted 50-fold pCAMBIA2300 plasmid, 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 primer 8 £0 10 ^^0: 18 and 8 £ 0 10 ^^0: 19 each of 2.0 ^, and 31 μΐ 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:
ACT^GCrJATGGTGGAGCACGACACTCT  ACT^GCrJATGGTGGAGCACGACACTCT
SEQ ID NO: 19：  SEQ ID NO: 19:
TGACJGC^GAGAGATAGATTTGTAGAGAGAGAC 使用引物 SEQ ID NO: 20和 SEQ ID NO: 21扩增 ThDH4 (模板是实施例 2所获得 的阳性 7¾DH¥-pGEM质粒），采用 stratagene的 PfuUltra II Fusion HS DNA Polymerase。 50 μ1 ΡΟ 反应体系： 5 μΐ lOxPfuUltra II reaction Buffer、 0.5μ1 25 mM的 dNTP、 2.0 μΐ ThDH4-pGEM质粒、 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ的引物 SEQ ID NO:20和 SEQ ID NO: 21各 2.0 μ1， 以及 37.5 μΐ的双蒸水。 PCR反应条件： 95 °C预 变性 2 min，35个循环（95 °C变性 25 s，51 °C退火 25 s，72°C延伸 30s)，72°C延伸 5 min。 通过 Pstl、 Sacl酶切后将所得 PCR产物连接 （连接方法同上） 到 pCAMBIA2300-3， 获得植物表达载体 35S-7¾DH¥-2300。 TGACJGC^GAGAGATAGATTTGTAGAGAGAGAC 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. 50 μl ΡΟ Reaction system: 5 μΐ lOxPfuUltra II reaction Buffer, 0.5 μl 25 mM dNTP, 2.0 μΐ ThDH4-pGEM plasmid, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ primers SEQ ID NO: 20 and SEQ ID NO: 21 Each of 2.0 μl, and 37.5 μΐ 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.
SEQ ID NO: 20：  SEQ ID NO: 20:
AACJGC^GATGGAGTCTAATTATCAAAACC  AACJGC^GATGGAGTCTAATTATCAAAACC
SEQ ID NO: 21：  SEQ ID NO: 21:
AAGGAGCTC CTAATAATGGCCACCGGGA 实施例 4 35S-ThDH4-2300表达载体转化农杆菌  AAGGAGCTC CTAATAATGGCCACCGGGA Example 4 35S-ThDH4-2300 Expression Vector Transformation Agrobacterium
农杆菌 LBA4404 (购自 Biovector Science Lab, Inc) 感受态细胞的制备： 提前 1-2 天将农杆菌 LBA4404在含 50 g/ml利福平和 50 g/ml链霉素的 LB固体培养基上划单斑 接种， 28°C培养 1至 2天。挑取单菌落接种于 5 ml含 50 μ§/ιη1利福平和 50 μ§/ιη1链霉素 的 LB液体培养基中， 28°C下摇动培养过夜 （约 12-16小时） 至 OD6(K)值为 0.4， 形成种 子菌液。取 5 ml活化后的菌液（ 1 :20的比例）接种于 100 ml含 50 g/ml利福平和 50 g/ml 链霉素的 LB液体培养基中， 28°C摇动培养 2-2.5小时至 OD6。。=0.8。 冰浴菌液 10 min, 每隔 3 min摇匀一次， 使细菌均匀进入休眠状态。 于 4°C下 4000 g离心 10 min， 弃上清 液； 加入一定量冰预冷的 10%甘油 （体积） 重悬浮菌体， 4°C下 4000 g离心 10 min， 收 集沉淀； 用冰预冷的 10%甘油 （体积） 重复洗 3-4次； 加入适量冰预冷的 10%甘油 （体 积） 重新悬浮细菌沉淀， 即制得 LBA4404感受态细胞， 以 40 μΐ/管将其分装， 于 -70°C保 存备用。 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 μ § /ιη1 rifampicin and 50 μ § /ιη1 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. 5 ml of activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, and cultured at 28 ° C for 2 to 2.5 hours with shaking. To OD 6 . . =0.8. The ice bath solution was shaken for 10 min every 3 min to allow the bacteria to enter the dormant state evenly. Centrifuge at 4000 g for 10 min at 4 °C, discard the supernatant; resuspend the cells by adding a certain amount of ice-cold 10% glycerol (volume), centrifuge at 4000 g for 10 min at 4 °C, collect the precipitate; Cold 10% glycerol (volume) was washed 3-4 times repeatedly; the bacterial pellet was resuspended by adding an appropriate amount of ice-cold 10% glycerol (volume) to prepare LBA4404 competent cells, which were dispensed at 40 μM/tube. Store at -70 ° C for later use.
转化农杆菌： 在冰上融化感受态细胞， 向 40 μΐ的感受态细胞中加入 1 μΐ实施例 3 中所得的阳性 35S-7 )H¥-2300 质粒， 混匀后冰浴约 10 min。 将所述感受态细胞和 35S-ThDH4-2300质粒 DNA的混合物用移液枪转移到冰预冷的电击杯（购自 bio-rad)中， 轻敲使悬浮液到达电击杯底部， 注意不要有气泡。 将电击杯放到电击室的滑道上， 推动 滑道将电击杯放至电击室基座电极处。 使用 0.1 cm 规格的电击杯， MicroPulser (购自 bio-rad)的程序设置为 "Agr"， 电击一次。立即取出电击杯，加入 28°C预热的 LB培养基。 快速而轻柔的用移液枪将感受态细胞打匀。将悬浮液转入 1.5 ml的离心管， 28°C， 225 rpm 摇动培养 1小时。 取 100-200 μΐ的菌液涂布于相应的抗性筛选培养基平板上（LB固体培 养基， 含 50 μ§/ιη1利福平、 50 g/ml链霉素和 50 g/ml卡那霉素）， 28°C培养。 筛选阳性 转化克隆， 并将其菌液于 -70°C保存备用。 实施例 5 利用农杆菌介导的转化法获得转基因拟南芥 Transformation of Agrobacterium: The competent cells were thawed on ice, and 1 μM of the positive 35S-7)H¥-2300 plasmid obtained in Example 3 was added to 40 μΐ 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. Using a 0.1 cm size electric shock cup, the MicroPulser (purchased from bio-rad) program 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. Quickly and gently mix the competent cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 hour with shaking. 100-200 μL of bacterial solution is applied to the corresponding resistant screening medium plate (LB solid culture) Nutrient, containing 50 μ § /ιη1 rifampicin, 50 g/ml streptomycin and 50 g/ml kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Obtaining transgenic Arabidopsis thaliana using Agrobacterium-mediated transformation
待转化植株培养： 拟南芥种子 （哥伦比亚型， 来自美国俄亥俄州立大学的拟南芥生 物资源中心） 播种在泥炭土中， 经 4°C低温处理 3天后， 置于 23 °C、 16小时光照 /8小时 黑暗的培养箱中发芽。 7-10天后移栽到装有泥炭土和蛭石（体积比 3: 1 ) 的口径为 7.5 cm 的塑料钵中， 每钵栽种 6株， 置于 23 °C， 16小时光照 /8小时黑暗的培养箱中生长。 移栽 前每钵浇营养液 1/2MS培养基（9.39 mM KN03， 0.625 mM KH2P04， 10.3 mM H4N03, 0.75 mM MgS04， 1.5 mM CaCl2， 50 μΜ ΚΙ, 100 μΜ Η3Β03， 100 M MnS04， 30 M ZnS04， 1 μΜ Na2Mo04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 μΜ FeS04) 40 ml, 移栽后视土 壤湿度及时补充水分。 在生长期间适当浇灌营养液。 按需要每 3-4周一次 （或者时间更 长）。 为了在每个植株上得到较多的花芽， 当大多数植株第一个花序形成后剪去第一个花 序， 解除顶端优势， 促使多个次生花序的同步出现。 当大多数花序约 1-10 cm高 （剪去 第一个花序后约 4-8天） 时准备浸染。 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. 1/2 MS 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 μΜ ΚΙ, 100 μΜ 钵 for each nutrient solution before transplanting. 3 Β0 3 , 100 M MnS0 4 , 30 M ZnS0 4 , 1 μΜ Na 2 Mo0 4 , 0.1 μΜ CoCl 2 , 100 μΜ Na 2 EDTA, 100 μΜ FeS0 4 ) 40 ml, the soil moisture is replenished in time after transplanting. The nutrient solution is properly watered during the growth period. Every 3-4 weeks (or longer) as needed. In order to obtain more flower buds on each plant, when the first inflorescence of most plants is formed, the first inflorescence is cut off, and the apical dominance is removed, prompting the simultaneous emergence of multiple secondary inflorescences. Dip is prepared when most inflorescences are about 1-10 cm high (about 4-8 days after the first inflorescence is cut).
农杆菌的培养： 取出实施例 4中保种的农杆菌阳性转化克隆的菌液活化后， 挑取农 杆菌单菌落接种到 10 mL无菌 LB液体培养基 （含 75 mg/L利福平、 100 mg/L链霉素和 100 mg/L卡那霉素）中， 28°C恒温下 250 r/min振摇过夜培养。再将所得到的菌液按 1%-2% 的体积比接种到 200 mL无菌 LB液体培养基（含 75 mg/L利福平、 100 mg/L链霉素和 100 mg/L卡那霉素） 中， 28°C下 250 171^^【亘温振摇使农杆菌的浓度达到006()()=1.8， 然后在 4 °。下 3000 r/min离心 15 min, 弃去上清液后用浸染培养基 （该浸染培养基是 1/2MS培 养基里加 5.0% (w/v) 的蔗糖和 0.05% ( 500 μΙ7ί) 的 Silwet L-77) 重新悬浮农杆菌， 悬 浮至 OD6QQ约 0.80。 Culture of 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). In the case of chloramphenicol, at 250 °C at 28 °C, the concentration of Agrobacterium reached 00 6 () () = 1.8, then at 4 °. Centrifuge at 3000 r/min for 15 min, discard the supernatant and use the dip medium (the dip medium is 5.0% (w/v) sucrose and 0.05% (500 μΙ7ί) of Silwet L in 1/2MS medium. -77) Resuspend Agrobacterium and suspend to OD 6 QQ about 0.80.
花序的浸染： 将上述含农杆菌的浸染培养基加入大口容器中， 每个口径 9 cm的容器 中加入 200-300 mL所述含农杆菌的浸染培养基用于浸染。 将植株倒置， 使地上组织全部 浸没在农杆菌悬浮液中 3-5 s， 并要轻轻搅动。 浸润后植株上应该有一层液体膜。 浸染过 的植株放在塑料盘中，用干净的塑料或保鲜膜覆盖以保湿，然后放置在弱光或暗处过夜， 注意小心防止阳光直射植株。 处理后约 12-24 小时去掉覆盖。 正常培养植株， 植株进一 步生长 3-5周， 直至角果变褐变干。 收获种子， 并将种子用离心管在 4 °C下干燥贮存。  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.
转基因种子筛选： 配制含 1/4 MS (4.695 mM KN03， 0.3125 mM KH2P04， 5.15 mM H4NO3, 0.375 mM MgS04， 0.75 mM CaCl2, 25 μΜ ΚΙ, 50 μΜ Η3ΒΟ3， 50 M MnSO4， 15 M ZnS04， 0.5 μΜ Να2Μο04, 0.05 M CoCl2， 50 μΜ Na2EDTA, 50 M FeSO4)大量 元素的水溶液，加入 0.8 % 琼脂粉，用微波炉加热至琼脂完全溶化，待冷却到 50°C 左右， 加入所需量的终浓度为 50 mg 的卡那霉素， 摇匀后每培养皿中倒入 25 mL， 置实验台 冷却凝固后即可播种。 把称量好的种子倒在一张普通复印纸上， 用手指轻敲复印纸， 将 种子均匀地播种在琼脂胶上， 盖上培养皿盖， 置 4 °C冰箱冷处理 72小时后， 移至 23 °C、 16小时光照 /8小时黑暗的培养箱中发芽， 定期统计种子发芽和幼苗生长情况， 将抗性幼 苗及时移栽到营养土中。移栽后视土壤湿度及时补充水分。在生长期间适当浇灌营养液。 取生长 20天的拟南芥叶片 0.1 g， 提取 DNA， 用 SEQ ID NO: 11： 禾 P SEQ ID NO: 12扩增 ThDH4 ， 50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ DNA、 1.0 μ1 Εχ Τα 10 μΜ的引物 SEQ ID NO: 11和 SEQ ID NO: 12 各 2.0 μ1， 以及 35 μΐ的双 蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环（94°C变性 45 s， 51 °C退火 45 s, 72°C 延伸 45 s)， 72°C延伸 7 min， 将 PCR鉴定为阳性的植株进行编号 （T1D1-T1D12), 并保 存。 实施例 6 过表达 ThDH4的转基因拟南芥 T1代植株的耐旱模拟实验及功能鉴定 灭过菌的蛭石用 1/2MS培养基浸透。 T1D1-T1D6及对照拟南芥种子分别播种在蛭石 上，每盆播种 10颗种子，25 °C、10小时光培养 /14小时暗培养循环，每 7天浇一次 1/2MS, 培养 20天之后， 每盆保留大小较一致的 4-5棵苗， 用于干旱实验。 转基因拟南芥、 对照 拟南芥干旱 14天 （不浇水）， 25 °C、 10小时光培养 /14小时暗培养循环。 T1代转基因植 株 （T0代转基因植株的种子长成的植株） 的抗旱性鉴定表明， 对照植株都萎蔫严重， 而 T1D1、 T1D2、 T1D3、 T1D4、 T1D5、 T1D6六个株系共 24棵 （每株系各 4-5棵） 拟南 芥中 21棵能够存活并继续生长，显现出明显的耐旱性（参见图 3a和 3b， 以 T1D2为例， T1D1、 T1D3、 T1D4、 T1D5、 T1D6 的结果与 T1D2类似， 在此未示出）。 实施例 7 在转录水平上验证 7 )H¥蛋白表达 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 μΜ ΚΙ, 50 μΜ Η 3 ΒΟ 3 , 50 M MnSO 4 , 15 M ZnS0 4 , 0.5 μΜ Να 2 Μο0 4 , 0.05 M CoCl 2 , 50 μΜ 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. Pour the weighed seeds onto a piece of plain copy paper, tap the copy paper with your fingers, evenly sow the seeds on the agar gel, cover the Petri dish, and cool the chamber at 4 °C for 72 hours, then move to Germinating in a 23 ° C, 16 hour light / 8 hour dark incubator, regular seed germination and seedling growth, and timely transplanting the resistant seedlings into the nutrient soil. After transplanting, the soil moisture is added to the water in time. The nutrient solution is properly watered during the growth period. 0.1 g of Arabidopsis leaves grown for 20 days, DNA was extracted, and ThDH4 was amplified with SEQ ID NO: 11: P SEQ ID NO: 12, 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ DNA, 1.0 μ1 Εχ Τα 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 μl, and 35 μΐ 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. Transgenic Arabidopsis thaliana, control Arabidopsis thaliana drought for 14 days (without watering), 25 °C, 10 hours light culture / 14 hours dark culture cycle. 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). 21 out of 4) Arabidopsis thaliana survived and continued to grow, showing obvious drought tolerance (see Figures 3a and 3b, using T1D2 as an example, T1D1, T1D3, T1D4, T1D5, T1D6 results and T1D2 is similar, not shown here). Example 7 Verification at the transcriptional level 7) H¥ protein expression
分别取对照拟南芥植株、 耐旱转基因拟南芥 T1 代植株 （分别属于 T1D1、 T1D2、 T1D3、 T1D4、 T1D5、 T1D6六个株系）、 和不耐旱转基因拟南芥 Tl代植株的干旱 10天 的叶片各 0.05 g， 用植物 RNA提取试剂盒 （Invitrogen)提取总 RNA。 用 HITACHI公司 的紫外分光光度计 U-2001测定总 RNA在 260 nm和 280 nm的吸光度值，计算各个 RNA 浓度。 依照 Invitrogen反转录试齐 Ll盒 Superscript III Reverse Transcriptase所示方法进行反 转录（2 g总 RNA作为模板，反转录引物为 SEQIDNO: 13)。通过 SEQIDNO:ll和 SEQ ID NO: 12扩增 T DH4， 检测 DH4蛋白相对表达情况。 Control Arabidopsis thaliana plants, drought-tolerant transgenic Arabidopsis thaliana T1 plants (six T1D1, T1D2, T1D3, T1D4, T1D5, T1D6, respectively), and drought-tolerant transgenic Arabidopsis thaliana T1 plants Total RNA was extracted with a plant RNA extraction kit (Invitrogen) by 0.05 g each for 10 days. The absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations. Inverted according to the Invitrogen reverse transcription test Ll box Superscript III Reverse Transcriptase Transcription (2 g total RNA as a template and reverse transcription primer as SEQ ID NO: 13). The relative expression of DH4 protein was detected by amplifying T DH4 by SEQ ID NO: 11 and SEQ ID NO: 12.
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶，以反转录的 cDNA为模板进行 PCR 反应。 50 μ1ΡΟ 反应体系： 10 μΐ 5xPS Buffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ cDNA 1.0 μΐ PrimeSTAR HS DNA聚合酶、 10 μΜ的引物 SEQ ID NO: 11禾 P SEQ ID NO: 12 各 2.0 μ1， 以及 30 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 29个循环（94°C 变性 45 s， 5 C退火 45 s， 72°C延伸 45s) ， 72°C延伸 10 min。  PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template. 50 μl ΡΟ Reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA 1.0 μΐ PrimeSTAR HS DNA polymerase, 10 μΜ primer SEQ ID NO: 11 and P SEQ ID NO: 12 each 2.0 μ1, and 30 ΐ ΐ 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.
产物电泳结果如图 4所示： M为 DNA Ladder Marker (DL2000, TakaRa) ， 1-5 为耐旱转基因拟南芥 T1代植株 （依次为： T1D1、 T1D2、 T1D3、 T1D4、 T1D5) ， 6-10为不耐旱转基因拟南芥 T1代植株， 11-14为非转基因拟南芥对照。图中所示 PCR 产物电泳条带大小与 J )H¥的大小一致 （约 380bp) 。 结果表明， 对照拟南芥没有 ThDH4转彔， 耐旱转基因拟南芥 T1代植株中 7¾DH¥的转录较强， 不耐旱转基因拟 南芥 T1代植株中 ThDH4的转录很弱。  The electrophoresis results of the product are shown in Figure 4: 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). The results showed that there was no ThDH4 switch in Arabidopsis thaliana, and the transcription of 73⁄4DH¥ in the T1 generation of drought-tolerant transgenic Arabidopsis thaliana plants was stronger, and the transcription of ThDH4 in the T1 generation plants of the drought-tolerant transgenic Arabidopsis thaliana was weak.

Claims

权 利 要 求 书 claims
1. 一个小盐芥脱水素编码基因编码的蛋白， 其氨基酸序列为 SEQ ID NO: 1。1. A protein encoded by the dehydrin-encoding gene of Salina chinensis, whose amino acid sequence is SEQ ID NO: 1.
2. 编码权利要求 1所述蛋白的基因， 其核苷酸序列如 SEQ ID NO: 2所示。 2. The gene encoding the protein of claim 1, whose nucleotide sequence is shown in SEQ ID NO: 2.
3. 一种重组表达载体， 其含有权利要求 2所述的基因并且所述基因的核苷酸序 列与所述表达载体的表达控制序列可操作地连接。 3. A recombinant expression vector, which contains the gene of claim 2 and the nucleotide sequence of the gene is operably linked to the expression control sequence of the expression vector.
4. 权利要求 3所述的重组表达载体，其为附图 2所示的 35S-7¾DH¥-2300载体。 4. The recombinant expression vector according to claim 3, which is the 35S-72DHY-2300 vector shown in Figure 2.
5. 一种重组细胞， 其含有权利要求 2所述的基因或者权利要求 3或 4所述的重 组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。 5. A recombinant cell containing the gene of claim 2 or the recombinant expression vector of claim 3 or 4; preferably, the recombinant cell is a recombinant Agrobacterium cell.
6. 一种改善植物耐旱性的方法， 包括： 将权利要求 2所述的基因或者权利要求 6. A method for improving plant drought tolerance, comprising: combining the gene described in claim 2 or the gene described in claim 2
3或 4所述的重组表达载体导入植物或植物组织并使所述基因表达； 优选地， 所述植 物是拟南芥。 The recombinant expression vector described in 3 or 4 is introduced into a plant or plant tissue and the gene is expressed; preferably, the plant is Arabidopsis thaliana.
7. 一种制备转基因植物的方法， 包括： 在有效产生植物的条件下培养含有权利 要求 2所述的基因或者权利要求 3或 4所述的重组表达载体的植物或植物组织。 7. A method for preparing transgenic plants, comprising: cultivating plants or plant tissues containing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions that are effective for producing plants.
8. 权利要求 7所述的方法， 其中所述植物是拟南芥。 8. The method of claim 7, wherein the plant is Arabidopsis thaliana.
9. 权利要求 2所述的基因、 权利要求 3或 4所述的重组表达载体或者权利要求 5所述的重组细胞用于改善植物耐旱性以及用于植物育种的用途。 9. The use of the gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 5 for improving plant drought tolerance and for plant breeding.
10. 权利要求 9所述的用途， 其中所述植物是拟南芥。 10. The use of claim 9, wherein the plant is Arabidopsis thaliana.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007005980A2 (en) * 2005-07-06 2007-01-11 Cornell Research Foundation Dehydrin genes and promoters from coffee
CN101955521A (en) * 2010-09-21 2011-01-26 中国农业大学 Plant stress tolerance associated protein, and coded genes and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007005980A2 (en) * 2005-07-06 2007-01-11 Cornell Research Foundation Dehydrin genes and promoters from coffee
CN101955521A (en) * 2010-09-21 2011-01-26 中国农业大学 Plant stress tolerance associated protein, and coded genes and application thereof

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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