WO2015042750A1 - 一种小盐芥脱水素蛋白dh6及其编码基因与应用 - Google Patents

一种小盐芥脱水素蛋白dh6及其编码基因与应用 Download PDF

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WO2015042750A1
WO2015042750A1 PCT/CN2013/001175 CN2013001175W WO2015042750A1 WO 2015042750 A1 WO2015042750 A1 WO 2015042750A1 CN 2013001175 W CN2013001175 W CN 2013001175W WO 2015042750 A1 WO2015042750 A1 WO 2015042750A1
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gene
seq
plant
expression vector
plants
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陈文华
孙超
崔洪志
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创世纪转基因技术有限公司
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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

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  • the present invention relates to plant proteins and their coding genes and applications, and in particular to a dehydrin protein derived from small salt mustard and a gene encoding the same, and the same in breeding Application in transgenic plants with improved drought. 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) (the gene designated herein as D, 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 D of small salt mustard (this article is named
  • ThDH6 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)H6-2300 vector shown in Figure 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)H6-2300) of T DH6 (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector C35S-7)H6-2300 of T DH6.
  • FIG. 3 shows the drought tolerance simulation results of 7) H6 T1 transgenic Arabidopsis plants (T1F6 in the figure) and non-transgenic Arabidopsis plants (Fig., 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: T1F1, T1F2, T1F3, T1F4, T1F5, T1F6, T1F7)
  • 8-12 is not drought tolerant
  • 13-16 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.
  • 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 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 ⁇ , and ligated overnight at 4 °C. Then 10 reaction products were 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 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) were placed in a 37 ° C shaker at 225 r/min.
  • Tryptone purchased from OXOID
  • yeast Extract purchased from OXOID
  • P 1% NaCl purchased from Sinopharm
  • the white colonies picked were inoculated into LB liquid medium containing 50 g/mL ampicillin in a 96-well cell culture plate (CORNING), and cultured overnight at 37 ° C, and glycerin was added to a final concentration of glycerol of 20% by volume. , kept at -80 ° C for use.
  • Nested PCR primer
  • Primer 1 and Primer 2R PCR-selectTM cDNA Subtraction Kit from Clontech
  • 166 positive clones were obtained, and all positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. Sequencing.
  • sequence was SEQ ID NO: 3. Sequence analysis indicated that the encoded protein of the sequence belonged to the dehydrin protein.
  • the full-length coding gene corresponding to the clone YLS-73 was named as 7).
  • H6, its corresponding protein is named Z)H6.
  • SEQ ID NO: 3 is the coding gene 73 ⁇ 4DH6
  • YLS-73GSP1 SEQ ID NO: 4:
  • GGCGTCGCTGTCTCAACCAC YLS-73GSP2 SEQ ID NO: 5:
  • AAACCCAGTCTCTTTGACAAGC 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-73GSP1 (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 tail of the Poly C tail to the 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 ⁇ 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 for 45 s at 94 °C, annealing for 45 s at 60 °C, extension of lmin at 72 °C), extension at 72 °C for 7 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 system 5 ⁇ ⁇ ⁇ ⁇ Buffer 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 ⁇ Ex Taq 10 ⁇ primer SEQ ID NO: 5 and P 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 57 °C for 45 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • a strip of about 750 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 (specifically the same as above), and 8 randomly selected.
  • the white colonies were inoculated separately in an LB liquid medium containing 50 ⁇ l of ampicillin, and 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).
  • SEQ ID NO: 10 is the full length sequence of 73 ⁇ 4 DH6.
  • a pair of primers were designed according to the sequence of SEQ ID NO: 10 as follows:
  • ThDH6F SEQ ID NO: 1 1:
  • ThDH6R SEQ ID NO: 12:
  • TTAAGCATCAGACTCTTTTTC AP SEQ ID NO: 13:
  • the 73 ⁇ 4DH6 full-length coding sequence was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
  • 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.
  • 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 50 °C for 25 s, extension at 72 °C for 40 s), 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 P 1.0 ⁇ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes.
  • the obtained DNA fragment of about 900 bp was recovered (Omega recovery kit), ligated into pGEM T-easy vector (to obtain ThDH6-pGEM plasmid), and then transformed into JM109, and 8 white colonies were randomly picked and inoculated to contain 50 g/
  • the mL ampicillin was cultured in LB liquid medium, and cultured at 37 ° C overnight, and then glycerin was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C for 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 ⁇ 4DH6 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 73 ⁇ 4DH6 gene, respectively, between the 35S 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 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 Kpnl and EcoRI and ligated into pCAMBIA2300-1 (Promega T4 ligase cassette) to obtain pCAMBIA2300-2.
  • SEQ ID NO: 16 CGGGG7MCCGAATTTCCCCGATCGTTCAAA
  • 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 ⁇ PS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ diluted 50-fold pCAMBIA2300 plasmid, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 18 and SEQ ID NO: 19 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 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 Xbal (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3
  • PCR reaction system 5 ⁇ lOxPfuUltra II reaction Buffer, 0.5 ⁇ 125 mM dNTP, 2.0 ⁇ ThDH6-pGEM plasmid, 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase 10 ⁇ primers SEQ ID NO: 20 and SEQ ID NO: 21 each 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 50 ° 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 Xbal and Kpnl (ligation method as above) to pCAMBIA2300-3 to obtain a plant expression vector 35S-73 ⁇ 4DH6-2300.
  • Agrobacterium tumefaciens LBA4404 (available from Biovector Science Lab, Inc) Preparation of competent cells: 1-2 in advance Agrobacterium tumefaciens LBA4404 was inoculated on a LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, and cultured 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)H6-2300 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-ThDH6-2300 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 ⁇ per ⁇ before transplanting. 3 , 100 M MnSO 4 , 30 M ZnSO 4 , 1 ⁇ Na 2 Mo0 4 , 0.1 ⁇ CoCl 2 , 100 ⁇ Na 2 EDTA, 100 ⁇ FeS0 4 ) 40 ml, and 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.
  • the first flower is cut off when the first inflorescence of most plants is formed. Preface, lifting the top advantage, 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).
  • 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%-2% to 200 mL sterile LB liquid culture (containing 75 mg/L rifampicin, 100 mg/L streptomycin and 100 mg/L Kanamycin).
  • 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 aboveground 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 them, and then placed in low light or dark places for the night, taking care to prevent direct sunlight from shining on 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 in a centrifuge tube at 4 °C.
  • 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 aqueous solution of the element, add 0.8% agar powder, and heat it in a microwave oven until the agar is completely dissolved.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 45 s, annealing at 50 °C for 45 s, extension at 72 °C for 45 s), extension at 72 °C for 7 min, identification of PCR as positive Plants are numbered (T1F1-T1F12), And save.
  • Example 6 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis thaliana T1 plants overexpressing T DH6 The sterilized vermiculite was soaked with 1/2 MS medium.
  • T1F1-T1F6 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 the T1 transgenic plants (plants grown from the seeds of the TO-transgenic plants) showed that the control plants were all wilted, and there were 24 strains of T1F1, T1F2, T1F3, T1F4, T1F5 and T1F6.
  • Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay (2 total RNA as a template and reverse transcription primer as SEQ ID NO: 13). The relative expression of DH6 protein was detected by amplifying T DH6 by SEQ ID NO: l l and SEQ ID NO: 12.
  • PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template.
  • 50 ⁇ l ⁇ Reaction system 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primer SEQ ID NO: 1 1 and P SEQ ID NO: 12 2.0 ⁇ 1 each , 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 50 °C for 45 s, extension at 72 °C for 45 s), extension at 72 °C for 10 min.
  • M is DNA Ladder Marker (DL2000, TakaRa)
  • 1-7 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant (in order: T1F1, T1F2, T1F3, T1F4, T1F5, T1F6, T1F7)
  • 8-12 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant
  • 13-16 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 73 ⁇ 4DH6 (about 900 bp).

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Abstract

本发明公开了一个来源于小盐芥的脱水素蛋白ThDH6、其编码基因，以及在培育耐旱性提高的转基因植物中的应用。

Description

种小盐芥脱水素蛋白 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 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
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。
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。
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系统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 D 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。
本发明第一方面提供小盐芥的一个脱水素蛋白 D 的编码基因 （本文命名为
ThDH6) ， 其序列为 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 )H6-2300载体。
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。
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。
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。
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。
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 DH6的植物表达载体 C35S-7 )H6-2300)的构建流程 （图 la-lb)。
图 2是 T DH6的植物表达载体 C35S-7 )H6-2300)的质粒图。
图 3是 7 )H6 T1代转基因拟南芥植株 （图中， T1F6) 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）。
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), 1-7为耐旱转基因拟南芥 T1代 植株（依次为： T1F1、 T1F2、 T1F3、 T1F4、 T1F5、 T1F6、 T1F7), 8-12为不耐旱转基因 拟南芥 Tl代植株， 13-16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。
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：
具体方法为：
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：
( 1 ) 供试材料：
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。
( 2 ) 材料处理：
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。
( 3 ) 总 RNA提取：
分别取对照组和干旱处理组的小盐芥叶片 0. 1 g， 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 polyA+ RNA from total RNA, 从总 RNA中纯化 polyA+ RNA)分离 mRNA。
( 4 ) 抑制差减杂交：
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。
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。
( 5 ) cDNA差减文库的构建与初步筛选、 克隆、 鉴定
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 μΐ ， 于 4°C连接过夜。 然后取 10 连接反应产物， 加入到 100 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 L 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。
( 6) 差异克隆的 cDNA测序分析：
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 ThDH6的克隆
克隆子 YLS-73去掉冗余 DNA后， 序列为 SEQ ID NO: 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-73对应的全长编码基因命名为 7 )H6， 其对应的蛋白命名为 Z)H6。
SEQ ID NO: 3
1 AAACCCAGTC TCTTTGACAA GCTTCACCGA TCCACCAGCT CTTCTTCCTC TTCAAGCGAT
61 GAAGAAGGTG AGGACGGTGA GAAGAGGAAG AAGAAGAAGG AGAAGAAGAA GACTGTCGAA 121 GGAGAGGATA AAACAGAGGA AGAGAATAAA GGAGTAATGG ACAAGATCAA GGAGAAGTTT 181 CCACACGCAA AGAAAACAGA GGATGATCAT GCACCAGTCG TCACCGGCGT CCCAGAGACG 241 GAGAAGATAG GAATGACCGA GAAGATAAAG GAGAAGCTTC CAGGCCACGG CAAGAAACCT 301 GAGGATTCAC CAGTCGTCGA CACCGCGCCG GTGGTTGAGA CAGCGACGCC AATTACGGCG 361 GAGCATTCGG CAGAGCATCC TGCGGAAAAG AAGGGATTTT TGCAAAAGAT CAAAGAAAAG 421 CTTCCAGGTC ATCACGCCAA GGGCACTGAA GAGATGGAGA AGAAAGAAAA AGAGTCTGAT 481 GCTTAA
DH6全长编码基因的克隆
根据已经获得的 SEQ ID NO: 3序列分析: SEQ ID NO: 3 为编码基因 7¾DH6的
3 ' 端序列。 根据已经获得的 SEQ ID NO: 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。
YLS-73GSP1 : SEQ ID NO: 4：
GGCGTCGCTGTCTCAACCAC YLS-73GSP2 : SEQ ID NO: 5：
TCGACGACTGGTGAATCCTC YLS-73GSP3 : SEQ ID NO:6:
AAACCCAGTCTCTTTGACAAGC 试剂盒自带通用引物：
AAP: SEQ ID NO: 7：
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）。
以 YLS-73GSP1(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 Poly C尾，以加尾后的产物为模板进行第一轮 PCR扩增，所用引物为 SEQ ID NO: 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：
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， 60°C退火 45 s， 72°C延伸 lmin)， 72°C延伸 7min。
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 SEQ ID NO: 8进行第二轮 PCR扩增， 具体步骤如下：
50 μΐ PCR反应体系： 5 μΐ ΙΟ Χ Εχ Buffer 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， 57°C退火 45 s， 72°C延伸 1 min)， 72°C延伸 10 min。回收第二次 PCR产物中约为 750bp 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 8个白色菌落分别接种于含有 50 ^lmL 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: 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。 所得的 5'RACE产物克隆子 06测序获得序列为 SEQ ID NO: 9： 1 GGGGGGGGGG ATGCTTTTTA AAATCACCTC AAAAAAGCAA TTAAAATCTC AATTCGAATT
61 TGAAATTTTC TCAGCTTTAT TTAATATTCT CGCTAGTCGA TCAAGAGTAA CAATGGCGGA
121 AGAGTACAAG AACGCTTCGG AGGAGTTCAA GAACGTTCCG GAACACGAGA CCACCCCAAA
181 GATCTCCACC ACGGAGGAAC CATCGGCAGA GGTTAAGGAT CGAGGATTTT TCGATTTCTT
241 GGGGAAGAAG AAAGAGGAAG TGAAACCTCA AGAGACCACG ACGCCACTCG AGTCTGAGTT
301 CGAGCACAAG GCTCAGATCT CGGAACCGCC GGCGTTTGTG GCGAAGCACG AAGAAGAGCA
361 AGAGACGAAG GAGAATAAGC CTACTCTCGT CGAGCAGCTT CACCAGAAAC ACGTGGAGGA
421 AGAACAGAAC AAACCCAGTC TCTTTGACAA GCTTCACCGA TCCACCAGCT CTTCTTCCTC
481 TTCAAGCGAT GAAGAAGGTG AGGACGGTGA GAAGAGGAAG AAGAAGAAGG AGAAGAAGAA
541 GACTGTCGAA GGAGAGGATA AAACAGAGGA AGAGAATAAA GGAGTAATGG ACAAGATCAA
601 GGAGAAGTTT CCACACGCAA AGAAAACAGA GGATGATCAT GCACCAGTCG TCACCGGCGT
661 CCCAGAGACG GAGAAGATAG GAATGACCGA GAAGATAAAG GAGAAGCTTC CAGGCCACGG
721 CAAGAAACCT GAGGATTCAC CAGTCGTCGA 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：
1 GGGGGGGGGG ATGCTTTTTA AAATCACCTC AAAAAAGCAA TTAAAATCTC AATTCGAATT
61 TGAAATTTTC TCAGCTTTAT TTAATATTCT CGCTAGTCGA TCAAGAGTAA CAATGGCGGA
121 AGAGTACAAG AACGCTTCGG AGGAGTTCAA GAACGTTCCG GAACACGAGA CCACCCCAAA
181 GATCTCCACC ACGGAGGAAC CATCGGCAGA GGTTAAGGAT CGAGGATTTT TCGATTTCTT
241 GGGGAAGAAG AAAGAGGAAG TGAAACCTCA AGAGACCACG ACGCCACTCG AGTCTGAGTT
301 CGAGCACAAG GCTCAGATCT CGGAACCGCC GGCGTTTGTG GCGAAGCACG AAGAAGAGCA
361 AGAGACGAAG GAGAATAAGC CTACTCTCGT CGAGCAGCTT CACCAGAAAC ACGTGGAGGA
421 AGAACAGAAC AAACCCAGTC TCTTTGACAA GCTTCACCGA TCCACCAGCT CTTCTTCCTC
481 TTCAAGCGAT GAAGAAGGTG AGGACGGTGA GAAGAGGAAG AAGAAGAAGG AGAAGAAGAA
541 GACTGTCGAA GGAGAGGATA AAACAGAGGA AGAGAATAAA GGAGTAATGG ACAAGATCAA
601 GGAGAAGTTT CCACACGCAA AGAAAACAGA GGATGATCAT GCACCAGTCG TCACCGGCGT
661 CCCAGAGACG GAGAAGATAG GAATGACCGA GAAGATAAAG GAGAAGCTTC CAGGCCACGG
721 CAAGAAACCT GAGGATTCAC CAGTCGTCGA CACCGCGCCG GTGGTTGAGA CAGCGACGCC
781 AATTACGGCG GAGCATTCGG CAGAGCATCC TGCGGAAAAG AAGGGATTTT TGCAAAAGAT
841 CAAAGAAAAG CTTCCAGGTC ATCACGCCAA GGGCACTGAA GAGATGGAGA AGAAAGAAAA
901 AGAGTCTGAT GCTTAA 根据 SEQ ID NO: 10序列分析， SEQ ID NO: 10 为 7¾DH6的全长序列。 根据 SEQ ID NO: 10 序列设计一对引物如下：
ThDH6F : SEQ ID NO: 1 1：
ATGCTTTTTAAAATCACCTCA
ThDH6R: SEQ ID NO: 12：
TTAAGCATCAGACTCTTTTTC AP : SEQ ID NO: 13：
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 通过 SEQ ID NO: 11和 SEQ ID NO: 12来克隆 7¾DH6全长编码序列。 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， 50°C退火 25 s， 72°C延伸 40s) ， 72°C延伸 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禾 P 1.0 μΐ Ex Taq。 反应条件： 70°C反应 30分钟。将得到的约 900bp的 DNA片段 回收（Omega回收试剂盒），连接至 pGEM T-easy载体上（得到 ThDH6-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
DH6蛋白的氨基酸序列： SEQ ID NO: 1
1 LFKITSKKQ LKSQFEFEIF SALFNILASR SRVTMAEEYK NASEEFK VP EHETTPKIST
61 TEEPSAEVKD RGFFDFLGKK KEEVKPQETT TPLESEFEHK AQISEPPAFV AKHEEEQETK
121 ENKPTLVEQL HQKHVEEEEN KPSLFDKLHR SSSSSSSSSD EEGEDGEKRK KKKEKKKTVE
181 GEDKTEEENK GV DKIKEKF PHAKKTEDDH APWTGVPET EKIG TEKIK EKLPGHGKKP
241 EDSPWDTAP WETATPITA EHSAEHPAEK KGFLEKIKEK LPGHHAKGTE E EKKEKESD
301 A
7¾DH6编码基因的核苷酸序列： SEQ ID NO: 2
1 ATGCTTTTTA AAATCACCTC AAAAAAGCAA TTAAAATCTC AATTCGAATT TGAAATTTTC
61 TCAGCTTTAT TTAATATTCT CGCTAGTCGA TCAAGAGTAA CAATGGCGGA AGAGTACAAG
121 AACGCTTCGG AGGAGTTCAA GAACGTTCCG GAACACGAGA CCACCCCAAA GATCTCCACC
181 ACGGAGGAAC CATCGGCAGA GGTTAAGGAT CGAGGATTTT TCGATTTCTT GGGGAAGAAG
241 AAAGAGGAAG TGAAACCTCA AGAGACGACG ACGCCACTCG AGTCTGAGTT CGAGCACAAG
301 GCTCAGATCT CGGAACCGCC GGCGTTTGTG GCGAAGCACG AAGAAGAGCA AGAGACGAAG
361 GAGAATAAGC CTACTCTCGT CGAGCAGCTT CACCAGAAAC ACGTGGAGGA AGAAGAGAAC
421 AAACCCAGTC TCTTTGACAA GCTTCACCGA TCCAGCAGCT CTTCTTCCTC TTCAAGCGAT
481 GAAGAAGGTG AGGACGGTGA GAAGAGGAAG AAGAAGAAGG AGAAGAAGAA GACTGTCGAA
541 GGAGAGGATA AAACAGAGGA AGAGAATAAA GGAGTAATGG ACAAGATCAA GGAGAAGTTT 601 CCACACGCAA AGAAAACAGA GGATGATCAT GCACCAGTCG TCACCGGCGT CCCAGAGACG
661 GAGAAGATAG GAATGACCGA GAAGATAAAG GAGAAGCTTC CAGGCCACGG CAAGAAACCT
721 GAGGATTCAC CAGTCGTCGA CACCGCGCCG GTGGTTGAGA CAGCGACGCC AATTACGGCG
781 GAGCATTCGG CAGAGCATCC TGCGGAAAAG AAGGGATTTT TGGAAAAGAT CAAAGAAAAG 841 CTTCCAGGTC ATCACGCCAA GGGCACTGAA GAGATGGAGA AGAAAGAAAA AGAGTCTGAT
901 GCTTAA 实施例 3 7¾£>H6基因植物表达载体构建
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启动子的上游插入 35S启动子及终 止子 Tnos分别作为 7¾DH6基因的启动子和终止子， 7¾DH6基因在所述 35S启动子和 Tnos终止子之间。
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 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: 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 ：
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 Kpnl、 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。
SEQ ID NO: 16： CGGGG7MCCGAATTTCCCCGATCGTTCAAA
SEQIDNO: 17：
TCKGAA rrCCC AGTGAATTCCCGATCT AGT A 使用引物 SEQIDNO: 18和 SEQIDNO: 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 μΙΡΟ 反应体系： 10 μΐ 5xPS 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、 10μΜ的引物 SEQIDNO:18和 SEQIDNO:19各 2.0μ1， 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 10min。 通过 HindIII、 Xbal酶切后将所得 PCR产物 连接到 （连接方法同上） pCAMBIA2300-2获得 pCAMBIA2300-3
SEQIDNO: 18：
ACT^GCrJATGGTGGAGCACGACACTCT
SEQIDNO: 19：
GC r ¾ AG AG AT AG ATTTGT AG AG AG AG AC 使用引物 SEQ ID NO: 20和 SEQ ID NO: 21扩增 ThDH6 (模板是实施例 2所获得 的阳性 7¾DH6-pGEM质粒），采用 stratagene的 PfuUltra II Fusion HS DNA Polymerase。 50 μΐ PCR反应体系： 5 μΐ lOxPfuUltra II reaction Buffer、 0.5μ125 mM的 dNTP、 2.0 μΐ ThDH6-pGEM质粒、 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ的引物 SEQ IDNO:20和 SEQIDNO: 21各 2.0 μ1， 以及 37.5 μΐ的双蒸水。 PCR反应条件： 95°C预 变性 2min，35个循环（95°C变性 25 s，50°C退火 25 s，72°C延伸 30s)，72°C延伸 5 min。 通过 Xbal、 Kpnl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-7¾DH6-2300。
SEQIDNO: 20：
AATCTAGAATGCTTTTTAAAATCACCTCA SEQIDNO: 21：
GCGGTACCTTAAGCATCAGACTCTTTTTC 实施例 4 35S-ThDH6-2300表达载体转化农杆菌
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。
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 )H6-2300 质粒， 混匀后冰浴约 10 min。 将所述感受态细胞和 35S-ThDH6-2300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
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 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 Η3ΒΟ3， 100 M MnSO4， 30 M ZnSO4， 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。
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 （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。
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。
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 ThDH6， 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， 50°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 （T1F1-T1F12), 并保存。 实施例 6 过表达 T DH6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。 T1F1-T1F6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 （TO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 T1F1、 T1F2、 T1F3、 T1F4、 T1F5、 T1F6 六个株系共 24棵 （每株系各 4-5棵） 拟南芥 中 20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 T1F6为例， T1F1、 T1F2、 T1F3、 TIF 4、 T1F5的结果与 T1F6类似， 在此未示出）。 实施例 7 在转录水平上验证 7 )H6蛋白表达
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 T1F1、T1F2、T1F3、 T1F4、 T1F5、 T1F6、 T1F7七个株系）、 和不耐旱转基因拟南芥 T1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 总 RNA作为模板，反转录引物为 SEQ ID NO: 13 )。通过 SEQ ID NO: l l和 SEQ ID NO: 12扩增 T DH6， 检测 DH6蛋白相对表达情况。
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 μ1 ΡΟ 反应体系： 10 μΐ 5 xPS 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: 1 1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， 50 °C退火 45 s， 72°C延伸 45s ) ， 72°C延伸 10 min。
产物电泳结果如图 4所示： M为 DNA Ladder Marker ( DL2000 , TakaRa) ， 1-7 为耐旱转基因拟南芥 T1代植株（依次为： T1F1、 T1F2、 T1F3、 T1F4、 T1F5、 T1F6、 T1F7 ) ， 8-12为不耐旱转基因拟南芥 Tl代植株， 13-16为非转基因拟南芥对照。 图 中所示 PCR产物电泳条带大小与 7¾DH6的大小一致 （约 900bp ) 。 结果表明， 对照 拟南芥没有 ThDH6转录， 耐旱转基因拟南芥 T1代植株中 ThDH6的转录较强， 不耐 旱转基因拟南芥 T1代植株中 ThDH6的转录很弱。

Claims

权 利 要 求 书
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。
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。
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。
4. 权利要求 3所述的重组表达载体，其为附图 2所示的 35S-7¾DH6-2300载体。
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。
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
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。
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。
8. 权利要求 7所述的方法， 其中所述植物是拟南芥。
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。
10. 权利要求 9所述的用途， 其中所述植物是拟南芥。
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