WO2015042739A1 - 一种小盐芥亮氨酸拉链蛋白bZIP-5及其编码基因与应用 - Google Patents

一种小盐芥亮氨酸拉链蛋白bZIP-5及其编码基因与应用 Download PDF

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WO2015042739A1
WO2015042739A1 PCT/CN2013/001158 CN2013001158W WO2015042739A1 WO 2015042739 A1 WO2015042739 A1 WO 2015042739A1 CN 2013001158 W CN2013001158 W CN 2013001158W WO 2015042739 A1 WO2015042739 A1 WO 2015042739A1
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plant
gene
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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Priority to CN201380078598.XA priority patent/CN105452279A/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

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  • the present invention relates to plant proteins and their coding genes and applications, and more particularly to a leucine zipper protein Z ⁇ 7-5 derived from small salt mustard and a gene encoding the same, and a transgenic plant thereof for improving drought tolerance Application in .
  • BACKGROUND OF THE INVENTION Stresses such as temperature, salting 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. Many regions are the bottleneck of agricultural development.
  • the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid regions account for 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 350-400 billion kilograms of grain; especially China's main grain-producing areas such as North China, Northeast China and Northwest China are the areas with the most water shortage in China, and the spring drought frequently reaches 10 years.
  • the present inventors used SSH (Suppression Subtractive Hybridization) in combination with RACE (Rapid Amplification of cDNA Ends)
  • SSH Stress Subtractive Hybridization
  • RACE Rapid Amplification of cDNA Ends
  • a leucine zipper protein of the small salt mustard was cloned (this article was named bZIP-5 encoding gene, and its DNA sequence was determined. It was found that after transduction into plants, the transgenic plants could be significantly improved. Drought tolerance, and these traits can be stably inherited.
  • the first aspect of the present invention provides a gene encoding a leucine zipper protein bZIP-5 of small salt mustard (herein named ThbZIP-5); preferably, the sequence thereof is SEQ ID NO: 2.
  • a second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into a basic vector for constructing the recombinant expression vector, And the nucleotide sequence of the gene is operably linked to the expression control sequence of the base vector; preferably, the base vector is pCAMBIA2300; preferably, the recombinant expression vector is -ThbZIP as shown in FIG. -5-2K vector.
  • 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 (358- ⁇ ) ⁇ / ⁇ -5-2300;) of ThbZIP-5 (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector ⁇ ;358- ⁇ ) ⁇ / ⁇ -5-2300 of ThbZIP-5.
  • FIG. 3 shows the results of drought tolerance simulation experiments of ⁇ ) ⁇ / ⁇ -5 T1 transgenic Arabidopsis plants (in the figure, T1M3) and non-transgenic Arabidopsis plants (in the figure, CK) as controls.
  • Fig. 3a is an Arabidopsis plant that is normally grown for 20 days
  • Fig. 3b is an Arabidopsis plant that has been subjected to drought treatment for 14 days after normal growth for 20 days.
  • Fig. 4 Results of changes in ABA content of T1 transgenic Arabidopsis plants and control plants under drought stress and normal growth conditions.
  • T1M1, T1M2, T1M3, T1M4, T1M5, T1M6, CK wherein T1M1, T1M2, T1M3, T1M4, T1M5, T1M6 are transgenic plants, and CK is a control plant.
  • FIG. 5 shows 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)
  • 1-4 is a drought-tolerant transgenic Arabidopsis thaliana T1 generation plant
  • 5-11 is a drought-tolerant transgenic Arabidopsis thaliana T1 generation plant (in order: T1M1, T1M2, T1M3, T1M4)
  • T1M5, T1M6, T1M7), 12-17 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 drought-treated salt mustard seedlings was used as a sample (Tester) during the experiment, and the mRNA of the leaves of the untreated small salt mustard seedlings was used as a control.
  • the specific steps are as follows:
  • TheUungie ha phila purchased from the Wulanbu and Desert Green Botanical Garden in the Bayanor City of Inner Mongolia, China, was planted on the vermiculite vermiculite at 25 °C, photoperiod 16 hours light / 8 Incubate under hours of darkness (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 ⁇ FeS0 4 ). It was used for experiments when the seedlings were as high as 25-30 cm.
  • the test seedlings were divided into 2 groups, 4 pots per group and 1 pot per pot.
  • the first group was a control group, cultured at 25 ° C, photoperiod of 16 hours light / 8 hours dark, and normal watering.
  • the second group was the drought treatment group, cultured at 25 °C, photoperiod 16 hours light/8 hours dark, stopped watering, treated for 10 days, and cut in time after treatment.
  • the leaves of the top 1/3 of the two seedlings were taken and rapidly frozen with liquid nitrogen and stored in a -70 °C refrigerator.
  • this experiment In order to increase the validity 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.
  • 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 (purchased from Promega kit) according to the procedure of the pGEM-T Easy kit
  • the vector is ligated, and the specific steps are as follows: The following components are sequentially added by using a 200 ⁇ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 ⁇ 1, 2 ⁇ 4 ligase buffer 5 , pGEM-T Easy vector 1 11, T4 DNA ligase 1 ⁇ , ligated overnight at 4 °C. Take 10 ligation reaction products, add to 100 competent E.
  • coli JM109 (purchased from TAKARA), ice bath for 30 minutes, heat shock for 60 seconds, ice bath for 2 minutes, and add 250 ⁇ L of LB medium (1% Tryptone from OXOID) , 0.5% Yeast Extract purchased from OXOID, 1% NaCl purchased from Sinopharm), placed in a 37 ° C water bath, shaken at 225 rpm for 30 minutes, take 200 ⁇
  • the bacterial solution was applied to LB (same as above)/X-gal/IPTG (X-gal/IPTG purchased from TAKARA, lg packaging) containing 50 g/mL ampicillin (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.).
  • the working concentration of 20 mg/ml mother liquor is: 200 ⁇ /100 ml LB medium above the mother liquor; IPTG is purchased from TaKaRa, and the 5g package is formulated into a mother liquor of 100 mM concentration.
  • the working concentration is: 100 ⁇ /lOOml of the above mother liquor LB culture Base) Incubate on solid culture plates for 18 hours at 37 °C. 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: Gh-B001 to Gh-B198).
  • the sequence was SEQ ID No: 3. Sequence analysis indicated that the encoded protein of the sequence belonged to the leucine zipper protein.
  • the full-length coding gene corresponding to the clone YLS-112 was named as ThbZIP-5, its corresponding protein is named 6Zff-5.
  • CAGCCAAATC AAAACAGCAT ATCATTCAAT GGCAATAA G ACTCCATGAT CTTGAATCAC
  • SEQ ID No: 3 is 3 encoding the gene bZIP-5 End sequence. Based on the sequence of SEQ ID NO: 3 that has been obtained, the following three specific primers were designed as reverse transcription primers and specific primers for 5 'RACE.
  • YLS-1 12GSP1 SEQ ID NO: 4
  • AGAAGTTGCTGCAACAGTGAC YLS-1 12GSP2 ( SEQ ID NO: 5 ) :
  • GATGGAGTTATTTGCAGCCC YLS-1 12 GSP3 ( SEQ ID NO: 6) :
  • the kit comes with universal primers:
  • AAP SEQ ID NO: 7 :
  • GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP SEQ ID NO: 8 :
  • the Ends kit was purchased from Invitrogen).
  • YLS-112GSP1 (SEQ ID NO: 4) as a reverse transcription primer, reverse transcription using small salt mustard mRNA as a template to obtain a cDNA template, and then adding a Poly C tail according to the procedure in the above 5' RACE kit instructions.
  • the first round of PCR amplification was carried out using the product after tailing as a template.
  • the primer used was SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (the kit is self-contained, I is a 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 minutes, 33 cycles (94 °C for 50 seconds, 58 °C for 50 seconds, 72 °C for 1 minute), 72 °C for 10 minutes.
  • the obtained PCR product was diluted 50-fold with double distilled water, and 2.0 ⁇ L was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 5 and the universal primer SEQ ID NO: 8.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 l Ex Taq, 10 ⁇ primers SEQ ID NO: 5 and SEQ ID NO: 8 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 50 seconds, annealing at 58 ° C for 50 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.
  • the primers SEQ ID NO: 5 and the 3' primer SEQ ID NO: 6 were used for PCR amplification (reaction system and reaction conditions as above), and 6 positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing. Sequencing, a 5' end sequence of the cDNA of the gene was obtained.
  • the obtained 5' RACE product clone YL16-3 was sequenced to obtain the sequence of SEQ ID NO: 9:
  • CTCGCTTTAT TCCTTAACAT TTGATGAGCT TCAGAACACA
  • CTAGGTGGAC CGGGGAAAGA 181 TTTCGGGTCG ATGAACATGG ATGAACTCCT GAAGAGCATA TGGACTGCTG AGGAAGCTCA
  • SEQ ID NO: 10 is 7) full length sequence of ⁇ / ⁇ -5 .
  • a pair of primers were designed according to the sequence of SEQ ID NO: 10 as follows:
  • ThbZIP-5F SEQ ID NO: 1 1 :
  • ThbZIP-5R SEQ ID NO: 12 :
  • ThbZIP-5 full-length coding sequence was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
  • the Arabidopsis thaliana RNA was extracted, and the primer SEQ ID NO: 13 was used as the reverse transcription primer to obtain the cDNA of the small salt mustard.
  • the PfuUltra II Fusion HS DNA Polymerase of Stratagene was used to carry out the PCR reaction using the cDNA of the small salt mustard as a template.
  • 50 ⁇ PCR reaction system 5 ⁇ lO 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: 11 and SEQ ID NO: 12 each 2.0 11, and 37.5 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 95 °C for 2 minutes, 35 cycles (denaturation at 95 °C for 25 seconds, annealing at 55 °C for 25 seconds, extension at 72 °C for 1 minute), extension at 72 °C for 5 minutes.
  • PCR amplification product plus A tail PCR product hydration to 400 ⁇ 1, first remove the protein with chloroform once, add the supernatant to add 3 ⁇ sodium acetate solution 40 ⁇ l, add 2 times 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, 1.0 l Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes.
  • a DNA fragment of about 1280 bp was recovered (Omega recovery kit), ligated into pGEM T-easy vector (7 >Z/P-5-pGEM plasmid), then transformed into JM109, and 6 white colonies were randomly picked and inoculated separately. Incubate in LB liquid medium containing 50 g/mL ampicillin, incubate at 37 °C overnight, add glycerol to a final concentration of 20% (v/v), and store at -80 °C until 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 4 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 protein encoded thereby is SEQ ID NO: 1.
  • Amino acid sequence of bZIP-5 protein SEQ ID NO: 1
  • the plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ⁇ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. .
  • the 35S promoter containing the double enhancer and the terminator Tnos were selected as promoters and terminators of the ThbZIP-5 gene, respectively.
  • 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, 10 ⁇ primers SEQ ID ⁇ : 14 and SEQ ID NO: 15 each 2.0 ⁇ l, and 31 ⁇ double Steamed water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes.
  • 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: 16 and P SEQ ID NO: 17 Amplification of Tnos using pBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase.
  • SEQ ID NO: 18 and SEQ ID NO: 19 were used to amplify the Arabidopsis thaliana 35S promoter 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, 10 ⁇ primer SEQ ID NO: 18 and P SEQ ID NO: 19 2.0 ⁇ l each, and 31 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, elongation at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was ligated by HindIII and Pstl (connection method is the same as above)
  • pCAMBIA2300-2 obtained pCAMBIA2300-3
  • TGACTGCAGAGAGATAGATTTGTAGAGAGAC SEQ ID NO: 20 and SEQ ID NO: 21 amplify ThbZIP-5 (template is positive for Example 2) rMZ/PJ-pGEM plasmid), Stratagene's PfuUltra II Fusion HS DNA Polymerase was used.
  • PCR reaction system 5 ⁇ lO PfuUltra II reaction Buffer, 0.5 ⁇ l 25 mM dNTP, 2.0 ⁇ ThbZIP-5-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 minutes, 35 cycles (denaturation at 95 ° C for 25 seconds, annealing at 57 ° C for 25 seconds, extension at 72 ° C for 45 seconds, extension at 72 ° C for 5 minutes. Digestion by Sall, Kpnl After the resulting PCR product is ligated (the connection method is the same as above) to lj
  • pCAMBIA2300-3 obtained the plant expression vector 35S-r)Z/P-5-2300.
  • 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 with shaking. The K) value is 0.4, and a seed bacterial liquid is formed.
  • Transformation of Agrobacterium Thaw competent cells on ice, add 1 ⁇ of the positive 35S-7M)Z/P-5-2300 plasmid obtained in Example 3 to 40 ⁇ of competent cells, mix and mix, and ice bath about 10 minute.
  • the mixture of competent cells and plasmid DNA was transferred to an ice-cold electric shock cup with a pipette, and tapped to bring the suspension to the bottom, taking care not to have air bubbles.
  • Place the electric shock cup (purchased from Bio-Rad) 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.
  • 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. 40 ml of nutrient solution per pot before transplanting, and the soil moisture should be replenished in time after transplanting. The nutrient solution is properly watered during the growth period.
  • Agrobacterium After removing the bacterial solution of the Agrobacterium-positive transformed clone preserved in Example 4, a single colony of Agrobacterium was picked and inoculated into 10 mL of sterile LB liquid medium (containing 75 mg/L rifampicin). 100 mg/L streptomycin and 100 mg/L kanamycin were shaken overnight at 250 rpm at 28 °C. Then, the obtained bacterial liquid was inoculated into 200 mL of LB liquid medium containing the above antibiotics at a ratio of 1% - 2% (v/v), and the concentration of Agrobacterium was reached at OD 6 (at a constant temperature of 28 ° C).
  • the infusion medium contains 5.0% (w/v) sucrose and 0.05% (500 ⁇ ⁇ Silwet L-77) resuspend Agrobacterium and suspend it to 0D 6QQ about 0.80.
  • 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 seconds and gently agitate. There should be a liquid film on the plant after infiltration. The immersed plants are placed in a plastic tray, covered with a clean plastic or plastic wrap to moisturize, and then placed in low light or dark overnight, taking care to prevent direct sunlight from the plant. 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. Seeds were harvested and the seeds were dried in a centrifuge tube at 4 °C.
  • PCR reaction conditions 94 °C pre-denaturation 5 Min, 33 cycles (45 °C for 45 s, 57 °C for 45 s, 72 °C for 1 min), 72 °C for 7 min), and plants identified as positive by PCR (T1M1-T1M12) And save.
  • T1M1-T1M12 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis thaliana T1 plants overexpressing ThbZIP-5. The sterilized vermiculite was soaked with 1/2 MS medium.
  • T1M1-T1M6 and control Arabidopsis seeds were planted on vermiculite, 10 seeds per pot, 25 °C, 10 hours light culture / 14 hours dark culture cycle, 1/2MS every 7 days, after 20 days of culture 4-5 seedlings of uniform size were kept in each pot for drought experiments.
  • the drought resistance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that the control plants were wilting, and there were 24 strains of T1M1, T1M2, T1M3, T1M4, T1M5 and T1M6.
  • ABA is recognized as a plant hormone associated with stress, which can act as a signaling molecule to regulate the expression of multiple stress-inducing genes, thereby improving the plant's resistance to stress.
  • T1M1, T1M2, T1M3, T1M4, T1M5, T1M6 was used under drought stress for 10 days and normal growth conditions, using phytohormone abscisic acid (ABA) enzyme.
  • the ELISA kit purchased from Shanghai Ruigu Biotechnology Co., Ltd. was used to determine the ABA content (see Figure 4).
  • RNA extracted by the plant RNA extraction kit was 0.05 g each of the leaves.
  • 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.
  • Reverse transcription (2 g total RNA as a template, reverse transcription primer SEQ ID NO: 13) was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase.
  • the relative expression of bZIP-5 protein was detected by amplifying ThbZIP-5 by SEQ ID NO: 11 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 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 ⁇ l, and 30 ⁇ Double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 minutes, 29 cycles (denaturation at 94 °C for 45 seconds, annealing at 55 °C for 45 seconds, extension at 72 °C for 1 minute), extension at 72 °C for 10 minutes.
  • M is the DNA Ladder Marker (DL2000, TakaRa)
  • 1-4 is the drought-tolerant transgenic Arabidopsis T1 plant
  • 5-1-1 is the drought-tolerant transgenic Arabidopsis thaliana T1 plant
  • 12-17 are non-transgenic Arabidopsis controls.
  • the size of the electrophoresis band of the PCR product shown in the figure is consistent with the size of 7)Z/P-5 (about 1280 bp).

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Abstract

提供了一种来源于小盐芥的亮氨酸拉链蛋白bZIP-5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。

Description

种小盐芥亮氨酸拉链蛋白 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亮 氨酸拉链蛋白 Z^7 -5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， 是许多地区是农业发展的瓶颈。 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， 许多植物对逆境下的生物化学和生理 学上的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 法克隆出了小盐芥的一种亮氨酸拉链蛋白 （本文命名为 bZIP-5 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种亮氨酸拉链蛋白 bZIP-5的编码基因 （本文命名为 ThbZIP-5) ； 优选地， 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， 其是 通过将所述基因插入到一种用于构建所述重组表达载体的基础载体而获得的， 并且所 述基因的核苷酸序列与所述基础载体的表达控制序列可操作地连接； 优选地， 所述基础 载体为 pCAMBIA2300; 优选地， 所述重组表达载体为附图 2所示的 -ThbZIP-5-2K 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 ThbZIP-5的植物表达载体 (358-Γ )Ζ/Ρ-5-2300;)的构建流程 （图 la-lb)。
图 2是 ThbZIP-5的植物表达载体 <;358-Γ )Ζ/Ρ-5-2300)的质粒图。
图 3是 Γ )Ζ/Ρ-5 T1代转基因拟南芥植株 （图中， T1M3 ) 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干旱胁迫和正常生长条件下的 T1代转基因拟南芥植株及对照植株 ABA含量 变化检测结果。 1-7依次为株系： T1M1、 T1M2、 T1M3、 T1M4、 T1M5、 T1M6、 CK， 其中 T1M1、 T1M2、 T1M3、 T1M4、 T1M5、 T1M6为转基因植株， CK为对照植株。
图 5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) , 1-4为不耐旱转基因拟南芥 T1 代植株， 5-11 为耐旱转基因拟南芥 T1 代植株 （依次为： T1M1、 T1M2、 T1M3、 T1M4、 T1M5、 T1M6、 T1M7 ) , 12-17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未注明来源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。 在实验过程中以干旱处理的盐芥幼苗的叶片的 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)。 具体步骤简述如 下：
( 1 ) 供试材料：
小盐芥 TheUungie ha phila, 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 μΜ FeS04) —次。 当苗株高达 25-30 cm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 1/3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 0.5 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 poly A+ RNA from total 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小时，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试剂盒的程序， 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 (购 自 Promega试剂盒)载体连接， 其具体步骤如下： 用 200 μΐ PCR管依次加入下列成分： 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 ，pGEM-T Easy载体 1 μ1， T4 DNA连接酶 1 μΐ ， 于 4°C连接过夜。 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分钟、 热休克 60秒、 冰 浴 2分钟， 另加 250 μL LB培养液 （ 1% Tryptone购自 OXOID, 0.5% Yeast Extract购自 OXOID， 1 % NaCl购自国药）置 37 °C水浴中， 以 225转 /分钟振荡培养 30分钟，取 200 μ 菌液涂布于含 50 g/mL氨苄青霉素 （购自天根生化科技 (北京)有限公司） 的 LB (同上） /X-gal/IPTG (X-gal/IPTG购自 TAKARA, lg包装配制成 20 mg/ml母液，其工作浓度为： 以上母液 200 μΙ/100 ml LB培养基； IPTG购自 TaKaRa, 5g包装配制成 100 mM浓度的母 液， 其工作浓度为： 以上母液 100 μΙ/lOOml LB培养基） 固体培养平板上， 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： Gh-B001至 Gh-B198)。 将所有白色克隆分别接种于含有 50 g/mL氨苄青霉素的 LB 液体培养基的 96 孔细胞培养板 (CORNING)中， 37°C培养过夜后加甘油至终浓度 20% (v/v), 于 -80°C保存备用。 以巢式 PCR 引物 Primer 1禾 P Primer 2R ( Clontech公司的 PCR-selectTM cDNA Subtraction Kit试剂盒自带）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在送英潍捷基 （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；)。 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 EST (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 盐芥亮氨酸拉链蛋白编码基因 ThbZIP-5的克隆
克隆子 YLS-112 去掉冗余 DNA后， 序列为 SEQ ID No: 3， 序列分析表明该序列的 编码的蛋白质属于亮氨酸拉链蛋白， 本文将克隆子 YLS-112对应的全长编码基因命名为 ThbZIP-5, 其对应的蛋白命名为 6Zff-5。
SEQ ID No: 3
CAGCCAAATC AAAACAGCAT ATCATTCAAT GGCAATAA G ACTCCATGAT CTTGAATCAC
61 CCACCTGGTT TAGGCCTCAA AGTGGG GGG GCAATGCAGC AGCAGCCACA ACAGCAGTTT
121 CAACAACAGC CACAACTGCA GTTGCAGCAG CAGCCACGAC AGCAGCTGAA TCAGCCACAT
181 CCACAGCAGC AGCAGCAGCG GCTGCCTCAG ACCATTTTTC CTAAACAAGC AAAGGTAGCA
241 TTGCTGGGC CTGTGGGTAT GGTCAACAAG AA TACGCCG GGGCTGCAAA TAACTCCATC
301 AACAATAATA ATGGATTAGC TAGT TCGGA GGAGCCGGGG TCACTGTTGC AGCAACTTCT
361 CCAGGGACAA GCAGCGCAGA AAACAATTCT TTGTCACCAG TTCTGTATGT GCTIAATCGA
421 GGAAGAAGAA GCAATAGGGG TCTAGAGAAG G TATCGAGA GGAGGCAAAG GAGAATGATC
481 AAGAACAGGG AATCAGCTGC TAGATCAAGA GCTCGAAAGC AGGCTTATAC CTTGGAACTG
541 GAAG TGAAA TTGAAAAGCT TAAGAAAGTG AACCAAGAAT TGCAGAGAAA GCAGGCTGAA
601 ATGA GGAAA TGCAGAAGAA TGAGCTGAAG GATCATCGAA GCAACCGTGG GGCAGCAAAA
661 GGCAATGCTT GAGAAGGACA CIAACCGGTC CATGGTAAAA TCA
bZIP-5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 bZIP-5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-1 12GSP1 ( SEQ ID NO: 4) ：
AGAAGTTGCTGCAACAGTGAC YLS-1 12GSP2 ( SEQ ID NO: 5 ) ：
GATGGAGTTATTTGCAGCCC YLS-1 12 GSP3 ( SEQ ID NO:6) ：
CAGCCAAATCAAAACAGCAT
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-112GSPl(SEQ ID NO: 4)为反转录引物，以小盐芥 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分钟， 33个循环（ 94 °C 变 性 50秒， 58°C退火 50秒， 72°C 延伸 1分钟）， 72 °C 延伸 10分钟。
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 Εχ Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ稀释的第一轮 PCR产物， 1.0 l Ex Taq、 10 μΜ的引物 SEQ ID NO: 5和 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分钟， 33个循环（ 94°C变性 50秒， 58°C 退火 50秒， 72°C延伸 1分钟）， 72 °C 延伸 10分钟。 回收第二次 PCR产物中约为 900bp 大小的条带 （Gel Extraction Kit购自 OMEGA), 并将其连接到 pGEM-T Easy Vector, 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 液体培养基中培养， 37°C培养过夜后加甘油至终浓度 20% (v/v), -80°C 下保存备用。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），得到 6个阳性克隆，送英潍捷基（上海）贸易有限公司测序测序， 获得该基因的 cDNA的一段 5'端序列。
所得的 5'RACE产物克隆子 YL16-3测序获得序列为 SEQ ID NO: 9：
1 GGGGGGGGGG ATGGGAACTC ACATCAATTT CAACAACTTA GGAGGTGGTG GTCCTCAGGG
61 AGGGGAAGGG AGTAACCAGA GAAGCCAAC GGGTAGTGCC ATTCCCTTGG CGAGGCAGTC 121 CTCGCTTTAT TCCTTAACAT TTGATGAGCT TCAGAACACA CTAGGTGGAC CGGGGAAAGA
181 T TCGGGTCG ATGAACATGG A GAACTCCT GAAGAGCATA TGGACTGCTG AGGAAGCTCA
241 GGCCATGGCC ATGACTTCTC CAGCTGTTAC AGCGGTAGCA CAACCTGG G GGAATCTCCA
301 GAGGCAAGGC TCGT GACAT TGCCTAGAAC GATTAGTCAG AAGACTGTTG ATGAGGTCTG
361 GAAA GTTTG ATCACCAAGG ACAGTAGGAA CGGTAATATG GGAGGTAGCA GCGGAGACGG 421 TGGTGAGTCG AATGCGCCTC CTGTGAGGCA ACAGACTTTA GGGGAGATGA CACTTGAAGA
481 ATTTCTGTTT CGTGCCGGAG TTGTTAGAGA AGATAACTGT ACTCAGCAGA TGGGTCAGGT
541 CAATGGAAAC AATAACAACG GGTITTA GG TAACAACGGC GCTGCTGGAG GATTAGGCTT
601 TGGGTTTGGT CAGCCAAATC AAAACAGCAT ATCATTCAAT GGCAATAA G ACTCCATGAT
661 CTTGAATCAC CCACCTGGTT TAGGCCTCAA AGTGGGTGGG GCAATGCAGC AGCAGCCACA 721 ACAGCAGTTT CAACAACAGC CACAACTGCA GTTGCAGCAG CAGCCACGAC AGCAGCTGAA
781 TCAGCCACAT CCACAGCAGC AGCAGCAGCG GCTGCCTCAG ACCATTTTTC CTAAACAAGC
841 AAACGTAGCA TTGCTGGGC CTGTGGGTAT GGTCAACAAG AATTACGCGG GGGCTGCAAA
901 TAACTCCATC 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 ATGGGAACTC ACATCAATTT CAACAACTTA GGAGGTGGTG GTCCTCAGGG
61 AGGGGAAGGG AGTAACCAGA TGAAGCCAAC GGGTAGTGCC ATTCCCTTGG CGAGGCAGTC
121 CTCGCTTTAT TCCTTAACAT TTGATGAGCT TCAGAACACA CTAGGTGGAC CGGGGAAAGA 181 TTTCGGGTCG ATGAACATGG ATGAACTCCT GAAGAGCATA TGGACTGCTG AGGAAGCTCA
241 GGCCATGGCC ATGACTTCTC CAGCTGTTAC AGCGGTAGCA CAACCTGGTG GGAATCTCCA
301 GAGGCAAGGC TCGT GACAT TGCCTAGAAC GATTAGTCAG AAGACTGTTG ATGAGGTCTG
361 GAAATGTTTG ATCACCAAGG ACAGTAGGAA CGGTAATATG GGAGGTAGCA GCGGAGACGG
421 TGGTGAGTCG AATGCGCCTC CTGTGAGGCA ACAGACTTTA GGGGAGATGA CACTTGAAGA 481 ATTTCTGTTT CGTGCCGGAG TTGTTAGAGA AGATAACTGT ACTCAGCAGA TGGGTCAGGT
541 CAATGGAAAC AATAACAACG GGTITTATGG TAACAACGGC GCTGCTGGAG GATTAGGCTT
601 TGGGTTTGGT CAGCCAAATC AAAACAGCAT ATCATTCAAT GGCAATAA G ACTCCATGAT
661 CTTGAATCAC CCACCTGGTT TAGGCCTCAA AGTGGGTGGG GCAATGCAGC AGCAGCCACA
721 ACAGCAGTTT CAACAACAGC CACAACTGCA GTTGCAGCAG CAGCCACGAC AGCAGCTGAA 781 TCAGCCACAT CCACAGCAGC AGCAGCAGCG GCTGCCTCAG ACCATTTTTC CTAAACAAGC
841 AAACGTAGCA TTTGCTGGGC CTGTGGGTAT GGTCAACAAG AATTACGCGG GGGCTGCAAA
901 TAACTCCATC AACAATAATA A GGATTAGC TAGT TCGGA GGAGCCGGGG TCACTGTTGC
961 AGCAACTTCT CCAGGGACAA GCAGCGCAGA AAACAATTCT TTGTCACCAG TTCGGTATGT
1021 GCTTAATCGA GGAAGAAGAA GCAATACGGG TCTAGAGAAG G TATCGAGA GGAGGCAAAG 1081 GAGAA GATC AAGAACAGGG AATCAGCTGC TAGATCAAGA GCTCGAAAGC AGGCTTATAC 1141 C TCGAACTG GAAGCCGAAA TTGAAAAGCT TAAGAAAGTG AACCAAGAAT TGCAGAGAAA 1201 GCAGGCTGAA A GATGGAAA TGCAGAAGAA TGAGCTGAAG GATCATCGAA GCAACCGTGG 1261 GGCAGCAAAA GGCAATGCTT GAGAAGGACA CTAACCGGTC CA GGTAAAA TCA 根据 SEQ ID NO: 10序列分析， SEQ ID NO : 10 为 7 )Ζ/Ρ-5的全长序列。 根据 SEQ ID NO: 10 序列设计一对引物如下：
ThbZIP-5F ( SEQ ID NO: 1 1 ) ：
ATGGGAACTCACATCAATTTC
ThbZIP-5R ( SEQ ID NO: 12 ) ：
TCAAGCATTG CCTTTTGCT AP ( SEQ ID NO: 13 ) ：
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 通过 SEQ ID NO: 11和 SEQ ID NO: 12来克隆 ThbZIP-5全长编码序列。
提取小盐芥 RNA， 以引物 SEQ ID NO: 13为反转录引物， 获取小盐芥 cDNA .采 用 Stratagene的 PfuUltra II Fusion HS DNA Polymerase, 以小盐芥的 cDNA为模板进行 PCR反应。 50 μΐ PCR反应体系： 5 μΐ lO 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: 11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分钟， 35个循环（95 °C 变性 25秒， 55 °C退火 25秒， 72°C 延伸 1分钟）， 72 °C 延伸 5分钟。
PCR扩增产物加 A尾： PCR产物补水至 400 μ1， 先用氯仿抽一遍去除蛋白， 吸取 上清加入 3Μ 醋酸钠溶液 40 μ1， 加 2倍的无水乙醇， -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 ΙΟχΕχ Buffer, 0.5 μΐ 5 mM的 dATP ， 1.0 l Ex Taq。 反应条件： 70°C反应 30分钟。 将得到约 1280bp的 DNA片段回收（Omega 回收试剂盒） ， 连接至 pGEM T-easy载体上 （得到 7 >Z/P-5-pGEM质粒） ， 然后转 化 JM109,随机挑取 6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终浓度 20% ( v/v) ， -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 4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。 bZIP-5蛋白的氨基酸序列： SEQ ID NO: 1
1 3THINENNL GGGGPQGGEG SNQVKPTGSA IPLARQSSVY SLTFDELQNT LGGPGKDFGS
61 M DELLKSI WTAEEAQAMA MTSPAVTAVA QPGG LQ QG SLTLPRTISQ K VDEVWKCL
121 ITKDSRNG GGSSGGGGES NAPPVRQQTL GEMTLEEFLF RAGWREDNC TQQ GQV GN 181 NNNGFYGNNG AAGGLGFGFG QPNQNSISFN GNNDSMID IQ PPGLGLKVGG AMQQQPQQQF
241 QQQPQLQLQQ QPRC3QLNQPH PQQQQQRLPQ TIFPKQANVA FAGPVGMVNK YAGAANNSI
301 NN NGLASPG GAGVTVAATS PGTSSAENNS LSPVPYVL R GRRSNTGLEK VIERRQRR I
361 KNRESAARSR AR QAY LEL EAEIEKLKKV NQELQRKQAE MVE QKNEL DHRSNRGAAK
421 GNA
7 )Z/P-5编码基因的核苷酸序列： SEQ ID NO: 2
1 ATGGGAACTC ACATCAATTT CAACAACTTA GGAGGTGG G GTCCTCAGGG AGGGGAAGGG
61 AGTAACCAGA TGAAGCCAAC GGGTAG GCC ATTCCCTTGG CGAGGCAGTC CTCGGI TAT
121 TCCTIAACAT TTGATGAGCT TCAGAACACA CTAGGTGGAC CGGGGAAAGA T TGGGGTCG
181 ATGAACATGG ATGAACTCCT GAAGAGCATA TGGACTGCTG AGGAAGCTCA GGCCATGGCC 241 ATGACTTCTC CAGCTGTTAC AGCGGTAGCA CAACCTGGTG GGAATCTCCA GAGGCAAGGC
301 TCG TGACAT TGCCTAGAAC GATTAGTCAG AAGACTGTTG ATGAGGTCTG GAAATGTTTG
361 ATCACCAAGG ACAGTAGGAA CGGTAATATG GGAGGTAGCA GCGGAGGCGG TGG GAGTCG
421 AATGGGCCTC CTGTGAGGCA ACAGACTTTA GGGGAGA GA CACTTGAAGA ATTTCTGTTT
481 CGTGCCGGAG TTGTTAGAGA AGATAACTGT ACTCAGCAGA TGGGTCAGGT CAA GGAAAC 541 AATAACAACG GGTTTTATGG TAACAACGGC GCTGCTGGAG GATTAGGCTT TGGGTTTGGT
601 CAGCCAAATC AAAACAGCAT ATCATTCAAT GGCAATAATG ACTCCATGAT CTTGAATCAG
661 CCACCTGGTT TAGGCCTCAA AGTGGGTGGG GCAATGCAGC AGCAGCCACA ACAGCAGTTT
721 CAACAACAGC CACAACTGCA GTTGCAGCAG CAGCCACGAC AGCAGCTGAA TCAGCCACAT
781 CCACAGCAGC AGCAGCAGCG GCTGCCTCAG ACCATTTTTC CTAAACAAGC AAAGGTAGCA 841 TTGCTGGGC CTGTGGGTAT GGTCAACAAG AA TACGCCG GGGCTGCAAA TAACTCCATC
901 AACAATAATA ATGGATTAGC TAGT TCGGA GGAGCCGGGG TCACTGTTGC AGCAACTTCT
961 CCAGGGACAA GCAGCGCAGA AAACAATTCT TTGTCACCAG TTCTGTATGT GCTIAATCGA
1021 GGAAGAAGAA GCAATACGGG TCTAGAGAAG G TATCGAGA GGAGGCAAAG GAGAATGATC
1081 AAGAACAGGG AATCAGCTGC TAGATCAAGA GCTCGAAAGC AGGCTTATAC CTTGGAACTG 1141 GAAGCGGAAA TTGAAAAGCT TAAGAAAG G AACCAAGAAT TGCAGAGAAA GCAGGCTGAA
1201 ATGATGGAAA TGCAGAAGAA TGAGCTGAAG GATCATCGAA GCAACCGTGG GGCAGCAAAA
1261 GGCAA GCTT GA 实施例 3 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 35S启动子， 以降低 ΝΡΤΠ蛋白在植物中的表达。 选择含双增强子的 35S启动子及终止子 Tnos分别作为 ThbZIP-5基因的启动子和终止子。
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、 10 μΜ的引物 SEQ ID ΝΟ: 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分钟， 33个循环（94°C 变性 30秒， 56°C退火 30 秒， 72°C 延伸 30秒）， 72 °C 延伸 10分钟。 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
SEQ ID NO: 16禾 P 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 ><PS Buffer, 3 μΐ 2.5 mM的 dNTP, 1.0 μΐ pBI121 , 1.0 μΐ Prime STAR、 10 μΜ的引物 SEQ ID NO: 16禾 P 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分钟， 33个循环 (94°C 变性 30秒， 58 °C退火 30秒， 72°C 延伸 30秒）， 72 °C 延伸 10分钟。通过 Sacl、 EcoRI 酶切后将所得 PCR 产物连接到 pCAMBIA2300-lCPromega T4 连接酶盒）获得 pCAMBIA2300-2。 SEQ ID NO: 16：
AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 17：
TCAGAATTCCCAGTGAATTCCCGATCTAGTA
SEQ ID NO: 18和 SEQ ID NO: 19以 pCAMBIA2300质粒为模板扩增拟南芥 35S 启动子。采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΙ ΡΟ 反应体系： 10 μΐ 5 ><PS Buffer, 3 μΐ 2.5 mM的 dNTP, 1.0 μΐ稀释 50倍的 pCAMBIA2300质粒， 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 18禾 P SEQ ID NO: 19各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR 反应条件： 94°C预变性 5分钟， 33个循环 (94°C变性 30秒， 50°C退火 30秒， 72°C延 伸 30秒）， 72°C延伸 10分钟。 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：
ACTAAGCTTATGGTGGAGCACGACACTCT SEQ ID NO: 19：
TGACTGCAGAGAGATAGATTTGTAGAGAGAGAC SEQ ID NO: 20和 SEQ ID NO: 21扩增 ThbZIP-5 (模板是实施例 2所获得的阳性 rMZ/P-J-pGEM质粒） ， 采用 Stratagene的 PfuUltra II Fusion HS DNA Polymerase。 50 μΐ PCR反应体系： 5 μΐ lO PfuUltra II reaction Buffer, 0.5μ1 25 mM的 dNTP， 2.0 μΐ ThbZIP-5-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分钟， 35个循环 (95 °C变性 25秒， 57°C退火 25秒， 72 °C延伸 45秒， 72°C延伸 5分钟。 通过 Sall、 Kpnl酶切后将所得 PCR产物连接 （连接方法同上） 至 lj
pCAMBIA2300-3 , 获得植物表达载体 35S-r )Z/P-5-2300。
SEQ ID NO: 20：
GCGTCGAC ATGGGAACTCACATCAATTTC SEQ ID NO: 21：
GGGGTACC TCAAGCATTG CCTTTTGCT 实施例 4 35S-ThbZIP-5-23 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同样浓度抗生素的 LB液体培养 基中， 28°C摇动培养 2-2.5小时至 OD6(K)=0.8。 冰浴菌液 10分钟， 每隔 3分钟摇匀一次， 使细菌均匀进入休眠状态。 于 4°C下 4000 g离心 10分钟， 弃上清液； 加入 10 ml冰预冷 的 10% (v/v)甘油重悬浮菌体， 4°C下 4000 g离心 10分钟， 收集沉淀； 用冰预冷的 10% (v/v) 甘油重复洗 3-4次； 加入 4 ml冰浴预冷的 10% (v/v) 甘油重新悬浮细菌沉淀， 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， 往 40 μΐ的感受态细胞中加入 1 μΐ实施例 3 中所得的阳性 35S-7M)Z/P-5-2300质粒， 混匀后冰浴约 10分钟。 将所述感受态细胞和质 粒 DNA的混合物用移液枪转移到冰预冷的电击杯中， 轻敲使悬浮液到达底部，注意不要 有气泡。 将电击杯（购自 Bio-Rad)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， MicroPuMUer (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 l ml LB培养基。 快速而轻柔的用移液枪 将细胞打匀。将悬浮液转入 1.5 ml的离心管，在 28°C下， 225 rpm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、 50 μ§/ιη1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 栽前每钵浇营养液 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 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液体培养基中 （含 75 mg/ L利福平、 100 mg/ L链霉素 和 100 mg/L卡那霉素）， 28°C恒温下 250转 /分钟振摇过夜培养。再将所得到的菌液按 1% - 2% (v/v) 的比例接种到 200 mL同样含上述抗生素的 LB 液体培养基中， 28°C恒温振 摇使农杆菌的浓度达到 OD6(K)=1.8， 然后在 4°C下 3000转 /分钟离心 15分钟， 弃去上清液 后用浸染培养基（该浸染培养基含有 5.0%(w/v)的蔗糖和 0.05%( 500 μΙΤθ的 Silwet L-77) 重新悬浮农杆菌， 悬浮至 0D6QQ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秒， 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 h后， 移至 23 °C、 16h光照 /8h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：和 SEQ ID NO: 12扩增 ThbZIP-5 ： ( 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 μΐ Ex Taq、 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， 57°C退火 45 s， 72 °C 延伸 1 min)， 72 °C 延伸 7 min)， 将 PCR鉴定 为阳性的植株进行编号 （T1M1-T1M12), 并保存。 实施例 6 过表达 ThbZIP-5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。 T1M1-T1M6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 T1M1、 T1M2、 T1M3、 T1M4、 T1M5、 T1M6 六个株系共 24棵 （每株系各 4-5棵） 拟 南芥中 22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 T1M3为例， T1M1、 T1M2、 T1M4、 TIC 5、 T1M6 的结果与 T1M3类似， 在此未示出）。 实施例 7干旱胁迫后 ABA变化的测定
ABA是公认的与逆境胁迫相关的一种植物激素， 可以作为信号分子调控多个逆境诱 导基因的表达， 从而提高植物的抗逆能力。 我们取干旱胁迫 10天和正常生长条件下的转 基因植株 （T1M1、 T1M2、 T1M3、 T1M4、 T1M5、 T1M6 ) 及对照植株 （CK1、 CK2 ) 叶片各 0.2 g左右， 用植物激素脱落酸 (ABA)酶联免疫（ELISA)试剂盒 (购自上海锐谷生 物科技有限公司） 测定 ABA含量 （见图 4)。 实验结果表明， 无论干旱处理还是对照条件 下， 转基因植株的 ABA含量均高于对照 （CK1、 CK2), 证明 Γ )Ζ/Ρ-5基因可以正调控 植物内源的 ABA含量。 实施例 8在转录水平上验证 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 T1M1、 T1M2、
T1M3、 T1M4、 T1M5、 T1M6六个株系）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，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 SEQ ID NO: 13 )。 通过 SEQ ID NO: 11和 SEQ ID NO: 12扩增 ThbZIP-5， 检测 bZIP-5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 10 μΜ的引物 SEQ ID NO: 16禾 Ρ SEQ ID NO: 17各 2.0 μ1， 以及 30 μΐ的双蒸水。 PCR反应条件： 94 °C预变性 5分钟， 29个循环 （94°C变性 45秒， 55 °C退火 45秒， 72°C延伸 1分钟） ， 72 °C延伸 10分钟。
产物电泳结果如图 5所示： M为 DNA Ladder Marker ( DL2000 , TakaRa) ， 1-4 为不耐旱转基因拟南芥 T1代植株， 5- 1 1为耐旱转基因拟南芥 T1代植株 （依次为： T1M1、 T1M2、 T1M3、 T1M4、 T1M5、 T1M6、 T1M7 ) ， 12-17为非转基因拟南芥对 照。 图中所示 PCR产物电泳条带大小与 7 )Z/P-5的大小一致 （约 1280bp ) 。 结果 表明， 对照拟南芥没有 7 )Z/P-5转录， 耐旱转基因拟南芥 T1代植株中 Γ )Ζ/Ρ-5的 转录较强， 不耐旱转基因拟南芥 T1代植株转录很弱。

Claims

权 利 要 求 书
1. 小盐芥的一种亮氨酸拉链蛋白， 其序列为 SEQ ID N0: 1。
2. 编码 SEQ ID NO: 1的亮氨酸拉链蛋白的基因，优选其序列为 SEQ ID NO: 2。
3. 一种重组表达载体， 其含有权利要求 2所述的基因插入到一种表达载体而获 得， 并且所述基因的核苷酸序列与用于构建所述重组表达载体的基础载体的表达控 制序列可操作地连接； 优选地， 所述基础载体为 pCAMBIA2300。
4. 权利要求 3所述的重组表达载体，其为附图 2所示的 35S-7 )Z/P-5-2300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 2或 3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 4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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Citations (1)

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CN101704884A (zh) * 2009-11-13 2010-05-12 北京市农林科学院 一种植物抗旱、耐盐相关蛋白EeABF6及其编码基因和应用

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CN101704884A (zh) * 2009-11-13 2010-05-12 北京市农林科学院 一种植物抗旱、耐盐相关蛋白EeABF6及其编码基因和应用

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