WO2014107863A1 - Cotton homeotic-leucine zipper protein hdbzip-1 and coding gene and use thereof - Google Patents

Cotton homeotic-leucine zipper protein hdbzip-1 and coding gene and use thereof Download PDF

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WO2014107863A1
WO2014107863A1 PCT/CN2013/070317 CN2013070317W WO2014107863A1 WO 2014107863 A1 WO2014107863 A1 WO 2014107863A1 CN 2013070317 W CN2013070317 W CN 2013070317W WO 2014107863 A1 WO2014107863 A1 WO 2014107863A1
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plant
seq
gene
expression vector
plants
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PCT/CN2013/070317
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French (fr)
Chinese (zh)
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陈文华
孙超
崔洪志
王君丹
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创世纪转基因技术有限公司
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Priority to CN201380069812.5A priority Critical patent/CN104968787A/en
Priority to PCT/CN2013/070317 priority patent/WO2014107863A1/en
Publication of WO2014107863A1 publication Critical patent/WO2014107863A1/en

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

Definitions

  • the present invention relates to plant proteins and coding genes thereof and applications thereof, and in particular to a cotton-derived homeotype-leucine zipper protein HDbZIP-1 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 areas account for about 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares.
  • Cubic meters due to lack of water, less than 350-40 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the most severe areas in China, and spring droughts frequently reach 10 years.
  • 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
  • HDbZIP homeobox-leucine zipper protein of cotton
  • the first aspect of the present invention provides a gene encoding a homeobox-leucine zipper protein HDbZIP-1 of cotton (designated herein as GhHDbZIP-1) having the sequence of SEQ ID NO: 2.
  • a second aspect of the invention provides a recombinant expression vector comprising the gene of the first aspect of the invention, and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, The vector is the 35S-GhHDbZIP-12-3300 vector shown in Fig. 2.
  • the third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
  • a fourth aspect of the present invention provides a method for improving drought tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and causing the gene Expression;
  • the plant is Arabidopsis thaliana.
  • a fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or a plant comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Tissue;
  • the plant is Arabidopsis thaliana.
  • a sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought tolerance of a plant and for use in plant breeding Use;
  • the plant is Arabidopsis thaliana.
  • the seventh aspect of the present invention provides the gene-encoded protein of the first aspect of the present invention, wherein the amino acid sequence thereof is
  • FIG. 1 is a construction flow of a plant expression vector (358-0111113 ⁇ 421?-1-2300;) of GhHDbZIP-1 (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector (35S-GhHDbZIP-1-23) of GhHDbZIP-1.
  • FIG. 3 shows the results of drought tolerance simulation experiments of GhHDbZIP-1 T1 transgenic Arabidopsis plants (in the figure, T1D2; T1D4) and non-transgenic Arabidopsis plants (Fig., CK1, CK2) 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 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.
  • 1-8 is the strain: T1D1, T1D2, T1D3, T1D4, T1D5, T1D6, CK1, CK2, Among them, T1D1, T1D2, T1D3, T1D4, T1D5 and T1D6 are transgenic plants, and CK1 and CK2 are control plants.
  • Figure 5 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 (DL5000, TakaRa), and 1-5 are drought-tolerant transgenic Arabidopsis T1 plants (in order: T1D1, T1D2, T1D3, T1D4, T1D5), 6-8 are non-transgenic Arabidopsis thaliana Control, 9-11 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant.
  • BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples.
  • Example 1 Construction of cotton SSH library under drought stress:
  • 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, photoperiod of 16 h light / 8 h dark, and was normally watered.
  • the second group was the drought treatment group, cultured at 25 °C, photoperiod 16h light/8h dark condition, stopped watering, and treated for 10 days. After the treatment, the leaves of the top 1/3 of the two seedlings were cut out in time, and the liquid nitrogen was quickly used. After freezing, store in a -70 ° C refrigerator.
  • the cotton leaves of the control group and the drought treatment group were respectively 0.5 g, and the total RNA of cotton leaves was extracted with a plant RNA extraction kit (purchased from invitrogen).
  • the absorbance of total RNA at 260 nm and 280 nm, OD 26 was determined using a HITACHI UV spectrophotometer U-2001. /OD 28 .
  • the ratio is 1.8-2.0, indicating total RNA High purity; total RNA integrity was detected by 1.0% agarose gel electrophoresis, and the 28S band was approximately twice as bright as the 18S band, indicating good RNA integrity.
  • mRNA was isolated using Qiagen's purification of poly A+ RNA from total RNA.
  • this experiment 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 (purchased from Promega kit) according to the procedure of the pGEM-T Easy kit
  • the vector is ligated as follows: The following components are sequentially added using a 200 ⁇ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 ⁇ 1, 2 ⁇ 4 ligase buffer 5 ⁇ l, pGEM-T Easy vector 1 ⁇ l, 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 min, heat shock for 60 s, ice bath for 2 min, and another 250 ⁇ L LB medium (1% Tryptone purchased from OXOID, 0.5% Yeast Extract was purchased from OXOID, 1% NaCl was purchased from Sinopharm. It was placed in a 37 °C water bath, shaken at 225 r/min for 30 min, and 200 ⁇ L of bacterial solution was applied to 50 g/mL ampicillin.
  • LB (ibid.) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA) was cultured on a plate at 37 ° C for 18 h.
  • Nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-select TM
  • the cDNA Subtraction Kit kit comes with PCR amplification of the bacterial cells, resulting in 216 positive clones, and then all positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • GhA137 GSP2 SEQ ID NO: 5:
  • GhA137 GSP3 SEQ ID NO: 6:
  • the kit comes with universal primers:
  • the experimental procedure was performed according to the kit instructions (3 'RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • 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: 8 each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (94 °C for 30 s, 56 °C for 30 s, 72 °C for 2 min), 72 °C for 10 min.
  • the obtained PCR product was diluted 50 times with double distilled water, and then 2.0 ⁇ L was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 5 and the universal primer SEQ ID NO: 8.
  • the specific steps were as follows: 50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 5 and SEQ ID NO: 8 each 2.0 ⁇ l, and 35 ⁇ 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 56 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • a strip of approximately 2.1 kbp in the second PCR product was recovered (Gel Extraction Kit was purchased from OMEGA) and ligated into pGEM-T Easy Vector, which was then transformed into E.
  • GhHD-2 GSP 1 SEQ ID NO: 10:
  • GhHD-2 GSP2 SEQ ID NO: 1 1:
  • GhHD-2 GSP3 SEQ ID NO: 12:
  • the kit comes with universal primers:
  • AAP SEQ ID NO: 13:
  • the GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIGGGIIG was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • GhHD-2 GSP1 (SEQ ID NO: 10) was used as a reverse transcription primer, and reverse transcription was performed using cotton mRNA as a template to obtain a cDNA template, and then Poly C tail was added according to the procedure in the above 5' RACE kit instructions. The tailed product was subjected to the first round of PCR amplification using the primer.
  • the primer used was SEQ ID NO: 10 and the universal primer SEQ ID NO: 13 (the kit is self-contained, and 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: 10 and SEQ ID NO: 13 each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min), 72 °C for 10 min.
  • the obtained PCR product was diluted 50-fold with double distilled water, and 2.0 ⁇ L was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 11 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: 11 and SEQ ID NO: 8 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (94 denaturation for 30 s, annealing at 53 °C for 30 s, 72V extension for 2 min), extension at 72 °C for 10 min.
  • a strip of approximately 900 bp in the second PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy Vector, then converted to JM109 (specifically the same as above), randomly picked 10 White colonies were inoculated separately in LB liquid medium containing 50 ⁇ ⁇ / ⁇ ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use.
  • PCR amplification of the bacterial solution using the primers SEQ ID NO: 11 and the 3' primer SEQ ID NO: 12 (reaction system and reaction) The conditions were the same as above.
  • GhHDbZIP-lF SEQ ID NO: 16:
  • GhHDbZIP-lR SEQ ID NO: 17:
  • the GhHDbZIP-1 full-length coding sequence was cloned by SEQ ID NO: 16 and SEQ ID NO: 17.
  • PCR was carried out using strafgene's PfUUltra II Fusion HS DNA Polymerase using cotton cDNA 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: 20 and SEQ ID NO: 21 each 2.0 ⁇ l, and 37.5 ⁇ M 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 54 °C for 30 s, extension at 72 °C for 1 min 30 s), extension at 72 °C for 5 min.
  • the PCR amplification product was added with A tail: The PCR product was added 2.5 times of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, and the supernatant was removed, dried, and dissolved in 21 ⁇ l of double distilled water. Add 2.5 ⁇ ⁇ ⁇ Buffer, 0.5 ⁇ 5 mM dATP, 1.0 ⁇ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 2.4 Kbp was recovered (Omega recovery kit), ligated into the pGEM T-easy vector (GhHDbZIP-1-pGEM plasmid was obtained), and then transformed into JM109 (method as above), and 8 white colonies were randomly picked.
  • the cells were inoculated in an LB liquid medium containing 50 g/mL ampicillin, cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20%, and stored at -80 ° C until use.
  • the primers SEQ ID NO: 16 and SEQ ID NO: 17 were used for PCR amplification (reaction system and reaction conditions are the same as above), and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • SEQ ID NO: 2 the amino acid sequence of the encoded protein is SEQ ID NO: 1
  • Nucleotide sequence of the GhHDbZIP-1 encoding 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 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 GhHDbZIP-1 gene, respectively.
  • Primer SEQ ID NO: 18 and SEQ ID NO: 19 were used to amplify Pnos 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 ⁇ primer SEQ ID 18: 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 56 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated into pCAMBIA2300 (promega, T4 ligase cassette) to obtain pCAMBIA2300-1.
  • SEQ ID NO: 20 and P SEQ ID NO: 21 Amplifies Tnos with pBI121 as a template, 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 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was digested with Sacl, EcoRI and ligated into the pCAMBIA2300-l Cpromega T4 ligase cassette) to obtain pCAMBIA2300-2.
  • SEQ ID NO: 22 and SEQ ID NO: 23 amplify the Arabidopsis thaliana 35S promoter using the pCAMBIA2300 plasmid as a template.
  • PrimeSTAR HS DNA polymerase using TaKaRa 50 ⁇ PCR reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ diluted 50-fold pCAMBIA2300 plasmid, 1.0 ⁇ PrimeSTAR, 10 ⁇ primer SEQ ID NO: 22 and P SEQ ID NO: 23 each 2.0 11, and 31 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (denaturation at 94 ° C for 30 s, annealing at 50 ° C for 30 s, Extend at 72 °C for 30 s;), extend at 72 °C for 10 min.
  • the resulting PCR product was ligated by HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3
  • GhHDbZIP-1 (template is the positive GhHDbZIP-1-pGEM plasmid obtained in Example 2), PfuUltra II Fusion HS DNA Polymerase using stratagene 50 ⁇ PCR reaction system: 5 ⁇ lO PfuUltra II reaction Buffer, 0.5 ⁇ l 25 mM dNTP, 2.0 ⁇ GhHDbZIP-l-pGEM plasmid, 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase, 10 ⁇ primer SEQ ID NO: 24 and P SEQ ID NO: 25 each 2.0 11, 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 58 °C for 30 s, extension at 72 °C for 1 min 30 s), extension at 72 °C for 5 min.
  • the resulting PCR product was ligated by BamHI and Sacl (ligation method) to pCAMBIA2300-3 to obtain a plant expression vector 35S-GhHDbZIP-l-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 in 5 1 ⁇ LB liquid medium containing 50 4 ⁇ /1 ⁇ rifampicin and 50 4 ⁇ /1 ⁇ streptomycin, and cultured overnight (about 12-16 h) at 28 °C. The OD 6QQ value was 0.4, and a seed bacterial liquid was formed.
  • Transformation of Agrobacterium The competent cells were thawed on ice, and 1 ⁇ of the positive 35S-GhHDbZIP-l-2300 plasmid obtained in Example 3 was added to 40 ⁇ of competent cells, and the mixture was mixed and ice bathed for about 10 min. 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.
  • the electric shock cup purchasedd from bio-rad
  • the MicroPuMUer purchased from bio-rad
  • the MicroPuMUer is set to "Agr" and the shock is applied once.
  • the electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added. Quickly and gently spread the cells with a pipette.
  • the suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h.
  • Plants to be transformed Arabidopsis seeds (Colombian type, Arabidopsis Bioresource Center, Ohio State University, USA) were sown in peat soil, treated at 4 ° C for 3 days, placed at 23 ° C, 16 h light / Germinated in a dark incubator at 8h. After 7-10 days, transplanted into a plastic pot with a diameter of 7.5 cm containing peat soil and vermiculite (3:1 by volume), 6 plants per pot, placed at 23 °C, 16h light/8h dark culture Growing in the box. 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 r/min at 28 °C.
  • 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 plant to make the ground tissue Immerse 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 plastic wrap to moisturize, then placed in low light or dark places overnight, taking care to prevent direct sunlight from the plants. The cover was removed about 12-24 hours after the treatment. 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.
  • Screening of transgenic seeds Prepare an aqueous solution containing 1/4 MS of large elements, add 0.8% agar powder, heat in a microwave oven until the agar is completely dissolved, and cool to about 50 °C, add the required amount of card with a final concentration of 50 mg 1 Natamycin, shake well, pour 25 mL into each dish, and set the table to cool and solidify before seeding.
  • PCR reaction conditions 94 °C predenaturation 5 Min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min), numbering plants identified as positive by PCR (T1D1-T1D16) And save.
  • T1D1-T1D16 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis thaliana T1 plants overexpressing GhHDbZIP-1. The sterilized vermiculite was soaked in 1/2 MS medium.
  • T1D1-T1D6 and control Arabidopsis seeds were planted on vermiculite, 10 seeds per pot, 25 ° C, 10 hours light culture / 14 hours dark culture cycle, 1/2 MS every 7 days, after 20 days of culture 4 seedlings of uniform size were kept in each pot for drought experiments.
  • the drought resistance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that the control plants were wilting, while T1D1, T1D2, T1D3, T1D4, T1D5, T1D6 had 24 strains (each line).
  • ABA is recognized as a plant hormone associated with stress, which can act as a signaling molecule to regulate multiple adverse temptations.
  • the expression of the derivative gene thereby improving the plant's resistance to stress.
  • the prepared kit measures the ABA content (see Figure 4). The results showed that the ABA content of transgenic plants was higher than that of the control (CK1, CK2) under drought treatment and control conditions, which indicated that GhHDbZIP-1 gene could positively regulate the endogenous ABA content of plants.
  • Example 8 Validation of GhHDbZIP protein expression at the transcriptional level
  • RNA extracted by a plant RNA extraction kit (invitrogen) at 0.05 g each for 10 days.
  • the absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations.
  • Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (2 ⁇ ⁇ total RNA as a template, reverse transcription primer SEQ ID NO: 7).
  • the relative expression of HDbZIP protein was detected by amplifying GhHDbZIP by SEQ ID NO: 16 and SEQ ID NO: 17.
  • 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 min, 29 cycles (denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • M is the DNA Ladder Marker (DL5000, TakaRa)
  • 1-5 is the drought-tolerant transgenic Arabidopsis T1 plant (in order: T1D1, T1D2, T1D3, T1D4, T1D5)
  • 6- 8 is a non-transgenic Arabidopsis control
  • 9-1 1 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant.
  • the size of the electrophoresis band of the PCR product shown in the figure is consistent with the size of GhHDbZIP-1 (about 2.4 Kbp).

Abstract

Provided are a plant protein and the coding gene and use thereof, in particular provided are a homeotic-leucine zipper protein GhHDbZIP-1 from cotton and the coding gene thereof, and use thereof for cultivating transgenic plants having improved drought tolerance.

Description

一种棉花同源异型-亮氨酸拉链蛋白 HDbZIP-1及其编码基因与应用  Cotton homeotype-leucine zipper protein HDbZIP-1 and its coding gene and application
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棉花的同源异 型-亮氨酸拉链蛋白 HDbZIP-1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。 FIELD OF THE INVENTION The present invention relates to plant proteins and coding genes thereof and applications thereof, and in particular to a cotton-derived homeotype-leucine zipper protein HDbZIP-1 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. According to statistics, the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid areas account for about 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares. Cubic meters, due to lack of water, less than 350-40 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the most severe areas in China, and spring droughts frequently reach 10 years.
植物耐旱性大多属于多基因控制的数量性状,利用常规育种方法改良作物的抗旱性受 到周期长、 优异种质资源缺乏的限制。 近年来的转录组学、 蛋白组学和基因表达调控的 研究初步揭示了植物干旱胁迫的作用分子机理。 目前，利用干旱胁迫相关基因提高植物的 抗旱能力,已经成为植物抗逆分子生物学的研究热点和植物抗逆基因工程重要的研究方 向。  Most of the drought tolerance of plants belongs to the quantitative traits controlled by multiple genes. The use of conventional breeding methods to improve the drought resistance of crops is limited by the long cycle and lack of excellent germplasm resources. Recent studies on transcriptomics, proteomics and gene expression regulation have revealed the molecular mechanism of plant drought stress. At present, the use of drought stress related genes to improve the drought resistance of plants has become a research hotspot of plant resistance to molecular biology and an important research direction of plant stress resistance genetic engineering.
植物受到逆境胁迫时会产生相应的应答反应， 以降低或消除逆境胁迫给植物带来的 危害。 植物的这种应答反应是一个涉及多基因、 多信号途径及多基因产物的复杂过程。 但就目前的研究状况而言， 由于其机制十分复杂，许多植物对逆境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  When plants are stressed by stress, they will respond accordingly to reduce or eliminate the damage caused by stress. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress remain to be further studied. Studies in the function and expression regulation of stress-responsive genes will provide an important basis for the link between plant stress-resistance-related signaling pathways and the study of the entire signaling network system. Summary of the invention
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 克隆出了棉花的一个同源异型-亮氨酸拉链蛋白 （本文命名为 HDbZIP) 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。 The present inventors used SSH (Suppression Subtractive Hybridization) in combination with RACE (Rapid Amplification of cDNA Ends) The coding gene of a homeobox-leucine zipper protein of cotton (herein named HDbZIP) was cloned and its DNA sequence was determined. Moreover, it was found that after being introduced into plants for over-expression, the drought tolerance of the transgenic plants can be significantly improved, and these traits can be stably inherited.
本发明第一方面提供棉花的一个同源异型-亮氨酸拉链蛋白 HDbZIP-1 的编码基因 (本文命名为 GhHDbZIP-1 ) ， 其序列为 SEQ ID NO: 2。  The first aspect of the present invention provides a gene encoding a homeobox-leucine zipper protein HDbZIP-1 of cotton (designated herein as GhHDbZIP-1) having the sequence of SEQ ID NO: 2.
本发明第二方面提供一种重组表达载体， 其含有本发明第一方面所述的基因并且所 述基因的核苷酸序列与所述表达载体的表达控制序列可操作地连接； 优选地， 所述载体 为附图 2所示的 35S-GhHDbZIP-l-2300载体。  A second aspect of the invention provides a recombinant expression vector comprising the gene of the first aspect of the invention, and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, The vector is the 35S-GhHDbZIP-12-3300 vector shown in Fig. 2.
本发明第三方面提供一种重组细胞， 其含有本发明第一方面所述的基因或者本发明 第二方面所述的重组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。  The third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
本发明第四方面提供一种改善植物耐旱性的方法， 包括： 将本发明第一方面所述的 基因或者本发明第二方面所述的重组表达载体导入植物或植物组织并使所述基因表达； 优选地， 所述植物是拟南芥。  A fourth aspect of the present invention provides a method for improving drought tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and causing the gene Expression; Preferably, the plant is Arabidopsis thaliana.
本发明第五方面提供一种制备转基因植物的方法， 包括： 在有效产生植物的条件下 培养含有本发明第一方面所述的基因或者本发明第二方面所述的重组表达载体的植物或 植物组织； 优选地， 所述植物是拟南芥。  A fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or a plant comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Tissue; Preferably, the plant is Arabidopsis thaliana.
本发明第六方面提供本发明第一方面所述的基因、 本发明第二方面所述的重组表达 载体或者本发明第三方面所述的重组细胞用于改善植物耐旱性以及用于植物育种的用 途； 优选地， 所述植物是拟南芥。  A sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought tolerance of a plant and for use in plant breeding Use; Preferably, the plant is Arabidopsis thaliana.
本发明第七方面提供本发明第一方面所述的基因编码的蛋白质， 其氨基酸序列如 The seventh aspect of the present invention provides the gene-encoded protein of the first aspect of the present invention, wherein the amino acid sequence thereof is
SEQ ID NO: 1所示。 附图说明 图 1是 GhHDbZIP-1的植物表达载体 (358-011111¾21?-1-2300；)的构建流程（图 la-lb)。 图 2是 GhHDbZIP-1的植物表达载体 (35S-GhHDbZIP-l-2300)的质粒图。 SEQ ID NO: 1 is shown. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector (358-0111113⁄421?-1-2300;) of GhHDbZIP-1 (Fig. la-lb). Figure 2 is a plasmid map of the plant expression vector (35S-GhHDbZIP-1-23) of GhHDbZIP-1.
图 3是 GhHDbZIP-1 T1代转基因拟南芥植株 （图中， T1D2; T1D4) 和作为对照的 非转基因拟南芥植株（图中， CK1、 CK2 ) 的耐旱模拟实验结果。 （图 3a为正常生长 20 天的拟南芥植株； 图 3b为正常生长 20天后干旱处理 14天的拟南芥植株）。  Figure 3 shows the results of drought tolerance simulation experiments of GhHDbZIP-1 T1 transgenic Arabidopsis plants (in the figure, T1D2; T1D4) and non-transgenic Arabidopsis plants (Fig., CK1, CK2) 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 20 days).
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。 1-8依次为株系： T1D1、 T1D2、 T1D3、 T1D4、 T1D5、 T1D6、 CK1、 CK2, 其中 T1D1、 T1D2、 T1D3、 T1D4、 T1D5、 T1D6为转基因植株， CK1、 CK2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 (DL5000,TakaRa) ， 1-5为耐旱转基因拟南芥 Tl 代植株 （依次为株系： T1D1、 T1D2、 T1D3、 T1D4、 T1D5 ) ， 6-8为非转基因拟南 芥对照， 9-11为不耐旱转基因拟南芥 T1代植株。 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。 实施例 1、 干旱胁迫下棉花 SSH文库构建： Fig. 4 Results of changes in ABA content of T1 transgenic Arabidopsis plants and control plants under drought stress and normal growth conditions. 1-8 is the strain: T1D1, T1D2, T1D3, T1D4, T1D5, T1D6, CK1, CK2, Among them, T1D1, T1D2, T1D3, T1D4, T1D5 and T1D6 are transgenic plants, and CK1 and CK2 are control plants. Figure 5 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 (DL5000, TakaRa), and 1-5 are drought-tolerant transgenic Arabidopsis T1 plants (in order: T1D1, T1D2, T1D3, T1D4, T1D5), 6-8 are non-transgenic Arabidopsis thaliana Control, 9-11 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples. Example 1. Construction of cotton SSH library under drought stress:
具体方法为：  The specific method is:
利用 Clontech公司的 PCR-selectTM 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棉花幼苗的叶片的 mRNA作为样本 (tester), 以未处理的棉花幼苗的叶片的 mRNA作为对照 （driver)。 具体步骤简述如下：The method of PCR-select TM cDNA Subtraction Kit from Clontech shown by using the subtractive hybridization method suppression construct subtraction libraries. The mRNA of the leaves of the drought-treated cotton seedlings was used as a tester during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a driver. The specific steps are as follows:
(1) 供试材料： (1) Test materials:
冀棉 14 (国家棉花中期库， 获取单位中国棉花研究所， 统一编号： ZM-30270) 播种到灭过菌的蛭石上， 在 25°C、 光周期 16h光照 /8h黑暗 （光强 2000— 3000 Lx) 条件下培养， 每周浇 1/2MS培养基 （9.39 mMKN03， 0.625 mM KH2P04， 10.3 mM NH4NO3, 0.75 mMMgSO4， 1.5 mMCaCl2， 50 μΜ KI， 100 μΜΗ3ΒΟ3， 100 MMnSO4， 30 MZnSO4， 1 μΜΝα2Μο04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 MFeSO4) — 次。 当苗株高达 25-30 cm时用于实验。 冀棉14 (National Cotton Medium-Term Library, obtained by the China Cotton Research Institute, Uniform No.: ZM-30270) Seeded onto the sterilized vermiculite, at 25 ° C, photoperiod 16h light / 8h dark (light intensity 2000-3000 Culture under conditions of Lx), 1/2 MS medium per week (9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM NH4NO3, 0.75 mM MgSO 4 , 1.5 mM CaCl 2 , 50 μΜ KI, 100 μΜΗ 3 ΒΟ 3 , 100 MMnSO 4 , 30 MZnSO 4 , 1 μΜΝα 2 Μο0 4 , 0.1 μΜ CoCl 2 , 100 μΜ Na 2 EDTA, 100 MFeSO 4 ) — times. It was used for experiments when the seedlings were as high as 25-30 cm.
(2) 材料处理：  (2) Material handling:
将供试幼苗分为 2组， 每组 4盆， 每盆 1株。 第一组为对照组， 在 25°C、 光周期 16h光照 /8h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 16h 光照 /8h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。  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, photoperiod of 16 h light / 8 h dark, and was normally watered. The second group was the drought treatment group, cultured at 25 °C, photoperiod 16h light/8h dark condition, stopped watering, and treated for 10 days. After the treatment, the leaves of the top 1/3 of the two seedlings were cut out in time, and the liquid nitrogen was quickly used. After freezing, store in a -70 ° C refrigerator.
(3) 总 RNA提取：  (3) Total RNA extraction:
分别取对照组和干旱处理组的棉花叶片 0.5 g， 用植物 RNA提取试剂盒 （购自 invitrogen) 提取棉花叶片的总 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， OD26。/OD28。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。 The cotton leaves of the control group and the drought treatment group were respectively 0.5 g, and the total RNA of cotton leaves was extracted with a plant RNA extraction kit (purchased from invitrogen). The absorbance of total RNA at 260 nm and 280 nm, OD 26 , was determined using a HITACHI UV spectrophotometer U-2001. /OD 28 . The ratio is 1.8-2.0, indicating total RNA High purity; total RNA integrity was detected by 1.0% agarose gel electrophoresis, and the 28S band was approximately twice as bright as the 18S band, indicating good RNA integrity. mRNA was isolated using Qiagen's purification of poly A+ RNA from total RNA.
( 4 ) 抑制差减杂交：  (4) Suppression of subtractive hybridization:
按 Clontech公司的 PCR-selectTM cDNA Subtraction Kit试剂盒所示的方法进行抑制差 减杂交。先将 Driver mRNA和 Tester mRNA分别反转录， 得到双链 cDNA, 再以 2 Tester cDNA禾 P 2 g Driver cDNA作为起始材料进行差减杂交。 在 37°C水浴下分别将 Tester cDNA和 Driver cDNA用 Rsa I酶切 1.5 h， 然后将酶切后的 Tester cDNA分成两等份， 连 接上不同的接头， 而 Driver cDNA不连接头。 两种连有不同接头的 Tester cDNA分别与 过量的 Driver cDNA混合， 进行第一次正向差减杂交。将两种第一次差减杂交的产物混合, 再与新变性的 Driver cDNA进行第二次正向差减杂交，然后通过两次抑制性 PCR扩增差 异表达的片段，使其得到富集。 Suppression Subtractive Hybridization performed by PCR-select TM cDNA Clontech's method shown Subtraction Kit kit. The Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and then subtracted hybridization using 2 Tester cDNA and P 2 g Driver cDNA as starting materials. The Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 h in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated. Two tester cDNAs with different adaptors were mixed with excess Driver cDNA for the first forward subtractive hybridization. The products of the two first subtractive hybridizations were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and then the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.
为了增加获得表达序列标签（Expressed sequence tag, EST) (unigene)的有效性， 避免 基因无酶切位点及所获得序列在非翻译区， 本实验同时用内切酶 Haelll 按上述步骤对 Tester cDNA和 Driver cDNA进行酶切并先后进行两次正向差减杂交和两次抑制性 PCR 扩增， 最后合并两组正向差减杂交 cDNA片段的第二次抑制性 PCR产物。  In order to increase the availability of Expressed sequence tag (EST) (unigene), avoid gene-free cleavage sites and the obtained sequences in the untranslated region, this experiment uses the endonuclease Haelll to tester cDNA according to the above steps. The cDNA was digested with Driver cDNA and subjected to two forward subtractive hybridizations and two inhibitory PCR amplifications. Finally, the second inhibitory PCR products of the two groups of forward subtractive hybridization cDNA fragments were combined.
( 5 ) cDNA差减文库的构建与初步筛选、 克隆、 鉴定  (5) Construction and preliminary screening, cloning and identification of cDNA subtraction library
依照 pGEM-T Easy试剂盒的程序， 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 μ1， 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 min、热休克 60 s、冰浴 2 min,另 力口 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 r/min振荡培养 30 min,取 200 μL菌液涂布于含 50 g/mL 氨苄青霉素的 LB (同上） /X-gal/IPTG ( X-gal/IPTG购自 TAKARA) 培养板上， 37°C培育 18 h。 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 266个白色菌落 (编 号： Gh-A001至 Gh-A266)。 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%, 于 -80°C保存备用。 以巢式 PCR 引物 Primer 1和 Primer 2R ( Clontech公司的 PCR-selectTM cDNA Subtraction Kit试剂盒自带） 进行菌液 PCR扩增， 得到 216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。 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 as follows: The following components are sequentially added using a 200 μΐ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 μ1, 2χΤ4 ligase buffer 5 μl, pGEM-T Easy vector 1 μl, T4 DNA ligase 1 μΐ, ligated overnight at 4 °C. Take 10 ligation reaction products, add to 100 competent E. coli JM109 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, and another 250 μL LB medium (1% Tryptone purchased from OXOID, 0.5% Yeast Extract was purchased from OXOID, 1% NaCl was purchased from Sinopharm. It was placed in a 37 °C water bath, shaken at 225 r/min for 30 min, and 200 μL of bacterial solution was applied to 50 g/mL ampicillin. LB (ibid.) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA) was cultured on a plate at 37 ° C for 18 h. Count the number of clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 266 white colonies (number: Gh-A001 to Gh-A266). All white clones were inoculated separately in 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 g/mL ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C. spare. Nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-select TM The cDNA Subtraction Kit kit comes with PCR amplification of the bacterial cells, resulting in 216 positive clones, and then all positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
(6) 差异克隆的 cDNA测序分析：  (6) cDNA sequencing analysis of differential clones:
将 DNA 测序结果去除载体和不明确序列及冗余的 cDNA 后， 共得到 173 个 EST(unigene)0 经分析有 27个重叠群， 有 146个单一的序列。 经 BlastN发现其中 85条 unigene在 GenBank 中有同源序列 （蛋白同源性 50%以上）， 29条 EST功能未知或者为 假定蛋白， 另有 32 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 棉花同源异型 -亮氨酸拉链蛋白编码基因 GhHDbZIP-1的克隆 After removing the carrier and DNA sequencing uncertainty of the cDNA sequence and the redundancy, to give a total of 173 EST (unigene) 0 analyzed contigs 27, 146 has a single sequence. It was found by BlastN that 85 unigenes have homologous sequences in GenBank (more than 50% protein homology), 29 ESTs are unknown or hypothetical proteins, and another 32 have not obtained homologous matches, presumably at 3' Or a shorter sequence of the untranslated region at the 5' end. Example 2 Cloning of a cotton homeobox-leucine zipper protein encoding gene GhHDbZIP-1
克隆子 Gh-A137去掉冗余 DNA后， 序列为 SEQ ID No: 3， 序列分析表明该序列的 编码的蛋白质属于同源异型 -亮氨酸拉链蛋白，本文将克隆子 Gh-A137对应的全长编码基 因命名为 GhHDbZIP-l， 其对应的蛋白命名为 HDbZIP-l。  After the cloned Gh-A137 was detached from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the encoded protein of the sequence belonged to the homeotype-leucine zipper protein, and the full length of the clone Gh-A137 was The coding gene was named GhHDbZIP-1 and its corresponding protein was named HDbZIP-1.
SEQ ID No: 3 SEQ ID No: 3
1 TCTATAAGAG ATGCAATGAG GAATCC A A TGTACTAATT GTGGTGGTCC GGCTATTATT 1 TCTATAAGAG ATGCAATGAG GAATCC A A TGTACTAATT GTGGTGGTCC GGCTATTATT
61 GGTGATATGT CACTTGAAGA ACAACTTCTT AGAATCGAAA ATGCTCGTTT AAAAGA GAA61 GGTGATATGT CACTTGAAGA ACAACTTCTT AGAATCGAAA ATGCTCGTTT AAAAGA GAA
121 TTAGATCGTG TTTGTGCACT TGCTGGTAAG TTTTTAGGTC GTCCAATTAC AGGACCTCCA121 TTAGATCGTG TTTGTGCACT TGCTGGTAAG TTTTTAGGTC GTCCAATTAC AGGACCTCCA
181 TTACCAAACT CAAGTTTAGA GGTTGGTGTT GGCACCAATG GTACTTTTGG AACCACTATG181 TTACCAAACT CAAGTTTAGA GGTTGGTGTT GGCACCAATG GTACTTTTGG AACCACTATG
241 GCTACTACTA CAACATTGCC TTTAGGACAT GATGCTTTAC CAACAATGGT TGTTCCTAGT241 GCTACTACTA CAACATTGCC TTTAGGACAT GATGCTTTAC CAACAATGGT TGTTCCTAGT
3 01 AATAGACCGG CAACAACACT CGATCGATCG ATGTTTTTGG AACTTGCTTT GGCTGCCATG3 01 AATAGACCGG CAACAACACT CGATCGATCG ATGTTTTTGG AACTTGCTTT GGCTGCCATG
361 GATGAACTTG TTAAGATGGC ACAAACTGAT GAGCCATTAT GGATTAAGAA CATAGAAGGT361 GATGAACTTG TTAAGATGGC ACAAACTGAT GAGCCATTAT GGATTAAGAA CATAGAAGGT
421 GGAAGAGAAA TGTTGAACCA TGATGAGTAT TTAAGGACAT TTACACCTTG TATTGGTTTA421 GGAAGAGAAA TGTTGAACCA TGATGAGTAT TTAAGGACAT TTACACCTTG TATTGGTTTA
481 AAACCAAATG GTTTTGTCAC TGAAGCGTCA AGGGAGACTG GTGTAGTGAT TA CAACAGT481 AAACCAAATG GTTTTGTCAC TGAAGCGTCA AGGGAGACTG GTGTAGTGAT TA CAACAGT
541 CTAGCCCTTG TTGAAACATT AGTGGACTCG AATCGTTGGG CAGAGATGTT TCATTGTATG541 CTAGCCCTTG TTGAAACATT AGTGGACTCG AATCGTTGGG CAGAGATGTT TCATTGTATG
601 ATTGC AGAA CATCAACAAC TGATGTGATA TCTAATGGCA TGGGAGGAAC CAGAAATGGT601 ATTGC AGAA CATCAACAAC TGATGTGATA TCTAATGGCA TGGGAGGAAC CAGAAATGGT
661 GCACTTCAAC A GAATGC TGAGCTTCAA ATCCTTTCAC CTTTAGTTCC TGTTCGTGAA661 GCACTTCAAC A GAATGC TGAGCTTCAA ATCCTTTCAC CTTTAGTTCC TGTTCGTGAA
721 GTAAGTTTCT TAAGGTTCTG TAAACAACAT GCTGAAGGTG TTTGGGCTGT GGTTGATGTA721 GTAAGTTTCT TAAGGTTCTG TAAACAACAT GCTGAAGGTG TTTGGGCTGT GGTTGATGTA
781 TCCGTTGATA C A CAAAGA AAGTACTACA TTTGTTACCT GTAGGAGACT TCCTTCTGGT781 TCCGTTGATA C A CAAAGA AAGTACTACA TTTGTTACCT GTAGGAGACT TCCTTCTGGT
841 TGTGTTGTTC AAGA ATGCC TAATGGTTAC TCCAAGGTTA TATGGGTTGA ACATGCT 841 TGTGTTGTTC AAGA ATGCC TAATGGTTAC TCCAAGGTTA TATGGGTTGA ACATGCT
HDbZIP-l全长编码基因的克隆 Cloning of the full-length coding gene of HDbZIP-1
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 3'RACE 的 5'端特异性引物。 GhA137 GSP1 : SEQ ID NO: 4： Based on the sequence of SEQ ID No: 3 that has been obtained, the following three specific primers were designed as the 5'-end specific primer for 3' RACE. GhA137 GSP1 : SEQ ID NO: 4:
TCTATAAGAGATGCAATGAGG  TCTATAAGAGATGCAATGAGG
GhA137 GSP2: SEQ ID NO: 5：  GhA137 GSP2: SEQ ID NO: 5:
ACTAATTGTGGTGGTCCGGC  ACTAATTGTGGTGGTCCGGC
GhA137 GSP3 : SEQ ID NO: 6：  GhA137 GSP3 : SEQ ID NO: 6:
AGGCATATCTTGAACAACAC  AGGCATATCTTGAACAACAC
试剂盒自带通用引物：  The kit comes with universal primers:
AP: SEQ ID NO: 7：  AP: SEQ ID NO: 7:
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT AUAP: SEQ ID NO: 8：  GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTT AUAP: SEQ ID NO: 8:
GGCCACGCGTCGACTAGTAC  GGCCACGCGTCGACTAGTAC
实验步骤按试剂盒说明书操作（ 3 ' RACE System for Rapid Amplification of cDNA Ends 试剂盒购自 invitrogen公司）。  The experimental procedure was performed according to the kit instructions (3 'RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
用 SEQ ID NO: 4与通用引物 SEQ ID NO: 8 (试剂盒自带的 AUAP引物），以 SEQ ID NO: 7弓 I物 （试剂盒自带的 AP弓 I物） 和棉花 mRNA反转录得到的 cDNA为模板进行第 一轮 PCR扩增。 具体步骤如下：  Using SEQ ID NO: 4 and the universal primer SEQ ID NO: 8 (the AUAP primer provided by the kit), reverse transcription of SEQ ID NO: 7 (the AP-specific I of the kit) and cotton mRNA The obtained cDNA was used as a template for the first round of PCR amplification. Specific steps are as follows:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ mRNA反转录 的 cDNA, 1.0 μΐ Ex Taq (购自 TAKARA)、 10 μΜ的引物 SEQ ID NO: 4和 SEQ ID NO: 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 30 s， 56°C退火 30 s， 72 °C 延伸 2 min)， 72 °C 延伸 10 min。  50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΐ Ex Taq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 4 and SEQ ID NO: 8 each of 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (94 °C for 30 s, 56 °C for 30 s, 72 °C for 2 min), 72 °C for 10 min.
所得的 PCR产物用双蒸水稀释 50倍后取 2.0 μΐ作为模板， 用 SEQ ID NO: 5与通用 引物 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 μΐ 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 min， 33个循环（ 94°C 变性 30 s， 56°C退火 30 s， 72 °C 延伸 2min)， 72 °C 延 伸 10 min。 回收第二次 PCR产物中约为 2.1 kbp大小的条带 （Gel Extraction Kit购自 OMEGA), 并将其连接到 pGEM-T Easy Vector, 然后转化到大肠杆菌 JM109 (具体方法同 上）， 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%， -80°C保存备用。 SEQ ID NO: 5与 3'端引物 SEQ ID NO: 6进行菌液 PCR扩增 （反应体系及条件同上） ， 得到 3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 3'端。 The obtained PCR product was diluted 50 times with double distilled water, and then 2.0 μL was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 5 and the universal primer SEQ ID NO: 8. The specific steps were as follows: 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 μΐ Ex Taq, 10 μΜ primers SEQ ID NO: 5 and SEQ ID NO: 8 each 2.0 μl, and 35 μΐ 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 56 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min. A strip of approximately 2.1 kbp in the second PCR product was recovered (Gel Extraction Kit was purchased from OMEGA) and ligated into pGEM-T Easy Vector, which was then transformed into E. coli JM109 (specifically as above), randomly picked Six white colonies were inoculated separately in LB liquid medium containing 50 g/mL ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. SEQ ID NO: 5 and the 3' primer SEQ ID NO: 6 were subjected to bacterial PCR amplification (reaction system and conditions are the same as above), and three positive clones were obtained, which were sent to English. Jieji (Shanghai) Trading Co., Ltd. sequenced and obtained the 3' end of the cDNA of the gene.
所得的 3'RACE产物克隆子 GhHD-2测序获得序列为 SEQ ID NO: 9：  The resulting 3' RACE product clone GhHD-2 was sequenced to obtain the sequence of SEQ ID NO: 9:
1 ACTAATTGTG GTGGTCCGGC TATTATTGGT GATATGTCAC TTGAAGAACA ACTTCTTAGA 1 ACTAATTGTG GTGGTCCGGC TATTATTGGT GATATGTCAC TTGAAGAACA ACTTCTTAGA
61 A CGAAAATG CTCGTTTAAA AGATGAAT A GATCGTGTTT GTGCACTTGC TGGTAAGTTT61 A CGAAAATG CTCGTTTAAA AGATGAAT A GATCGTGTTT GTGCACTTGC TGGTAAGTTT
121 TTAGGTCGTC CAATTACAGG ACCTCCATTA CCAAACTCAA GTTTAGAGCT TGGTGTTGGC121 TTAGGTCGTC CAATTACAGG ACCTCCATTA CCAAACTCAA GTTTAGAGCT TGGTGTTGGC
181 ACCAATGGTA CTTTTGGAAC CACTATGGCT ACTACTACAA CATTGCCTTT AGGACATGAT181 ACCAATGGTA CTTTTGGAAC CACTATGGCT ACTACTACAA CATTGCCTTT AGGACATGAT
241 GCTTTACCAA CAATGGTTGT TCCTAGTAAT AGACCGGCAA CAACACTCGA TCGATCGATG241 GCTTTACCAA CAATGGTTGT TCCTAGTAAT AGACCGGCAA CAACACTCGA TCGATCGATG
3 01 TTTTTGGAAC TTGCTTTGGC TGCCATGGAT GAACTTGTTA AGATGGCACA AACTGATGAG3 01 TTTTTGGAAC TTGCTTTGGC TGCCATGGAT GAACTTGTTA AGATGGCACA AACTGATGAG
361 CCATTATGGA TTAAGAACAT AGAAGGTGGA AGAGAAATGT TGAACCATGA TGAGTATTTA361 CCATTATGGA TTAAGAACAT AGAAGGTGGA AGAGAAATGT TGAACCATGA TGAGTATTTA
421 AGGACATTTA CACCTTGTAT TGGTTTAAAA CCAAATGGTT TTGTCACTGA AGCGTCAAGG421 AGGACATTTA CACCTTGTAT TGGTTTAAAA CCAAATGGTT TTGTCACTGA AGCGTCAAGG
481 GAGACTGGTG TAGTGATTAT CAACAGTCTA GCCCTTGTTG AAACATTAAT GGACTCGAAT481 GAGACTGGTG TAGTGATTAT CAACAGTCTA GCCCTTGTTG AAACATTAAT GGACTCGAAT
541 CGTTGGGCAG AGATGTTTCA TTGTATGATT GCTAGAACAT CAACAACTGA TGTGATATCT541 CGTTGGGCAG AGATGTTTCA TTGTATGATT GCTAGAACAT CAACAACTGA TGTGATATCT
601 AATGGCATGG GAGGAACCAG AAATGGTGCA CTTCAACTTA TGAATGCTGA GCTTCAAATC601 AATGGCATGG GAGGAACCAG AAATGGTGCA CTTCAACTTA TGAATGCTGA GCTTCAAATC
661 CTTTCACCTT TAGTTCCTGT TCGTGAAGTA AGTTTCTTAA GGTTCTGTAA ACAACATGCT661 CTTTCACCTT TAGTTCCTGT TCGTGAAGTA AGTTTCTTAA GGTTCTGTAA ACAACATGCT
721 GAAGGTGTTT GGGCTGTGGT TGATGTATCC GTTGATACTA TCAAAGAAAG TACTACATTT721 GAAGGTGTTT GGGCTGTGGT TGATGTATCC GTTGATACTA TCAAAGAAAG TACTACATTT
781 GTTACCTGTA GGAGACTTCC TTCTGGTTGT GTTGTTCAAG ATATGCCTAA TGGTTACTCC781 GTTACCTGTA GGAGACTTCC TTCTGGTTGT GTTGTTCAAG ATATGCCTAA TGGTTACTCC
841 AAGGTTATAT GGGTTGAACA TGCTGAA AT GATGAGAGCC AAGTTCATCA ACTATACCGG841 AAGGTTATAT GGGTTGAACA TGCTGAA AT GATGAGAGCC AAGTTCATCA ACTATACCGG
901 CCTTTACTAA GTTCCGGCGT GGGCTTCGGT GCCCAACGGT GGGTGGCGGC CCTTCAACGG901 CCTTTACTAA GTTCCGGCGT GGGCTTCGGT GCCCAACGGT GGGTGGCGGC CCTTCAACGG
961 CAATGCGAAT GCCTTGCCAT ACTCATGTCC TCCACCGTTC CCACTAGAGA CCACACCGCT961 CAATGCGAAT GCCTTGCCAT ACTCATGTCC TCCACCGTTC CCACTAGAGA CCACACCGCT
1021 ATAACTGCGA GTGGGAGACG GAGCATGTTA AAGCTAGCTC AACGGATGAC AGATAACTTC1021 ATAACTGCGA GTGGGAGACG GAGCATGTTA AAGCTAGCTC AACGGATGAC AGATAACTTC
1081 TGTGCCGGGG TTTGTGCATC GACAGTTCAT AAATGGAACA AGCTTAACGC CGGGAATGTA1081 TGTGCCGGGG TTTGTGCATC GACAGTTCAT AAATGGAACA AGCTTAACGC CGGGAATGTA
1141 GACGAAGACG TTAGGGTTAT GACTAGGAAA AGCATTGACG ACCCCGGCGA ACCGCCAGGG1141 GACGAAGACG TTAGGGTTAT GACTAGGAAA AGCATTGACG ACCCCGGCGA ACCGCCAGGG
12 01 ATCGTACTAA GCGCCGCCAC TTCGGTTTGG TTGCCGGTGT CACCGCAACG GCTTTTCGAT12 01 ATCGTACTAA GCGCCGCCAC TTCGGTTTGG TTGCCGGTGT CACCGCAACG GCTTTTCGAT
1261 TTCCTACGCG ACGAACGGTT GAGGAGCGAG TGGGACATAT TGTCAAATGG CGGACCTATG1261 TTCCTACGCG ACGAACGGTT GAGGAGCGAG TGGGACATAT TGTCAAATGG CGGACCTATG
1321 CAAGAGATGG CACACATTGC CAAAGGCCAA GATCATGGCA ATTGCGTTTC CCTCCTGCGT1321 CAAGAGATGG CACACATTGC CAAAGGCCAA GATCATGGCA ATTGCGTTTC CCTCCTGCGT
1381 GCCAGTGCCA TGAACGCAAA TCAGAGCAGC ATGCTGATAT TGCAGGAAAC ATGCATAGAC1381 GCCAGTGCCA TGAACGCAAA TCAGAGCAGC ATGCTGATAT TGCAGGAAAC ATGCATAGAC
1441 GCAGCAGGGT CGCTTGTAGT GTACGCGCCA GTTGATATTC CAGCCATGCA CGTTGTCATG1441 GCAGCAGGGT CGCTTGTAGT GTACGCGCCA GTTGATATTC CAGCCATGCA CGTTGTCATG
1501 AACGGTGGTG ATTCCGCTTA CGTCGCACTT TTACCTTCGG GATTCGCCAT TGTCCCGGAC1501 AACGGTGGTG ATTCCGCTTA CGTCGCACTT TTACCTTCGG GATTCGCCAT TGTCCCGGAC
1561 GGTCCTGGGT CTCATGGACC TATCTCTAAC GGACATGTTA ACGGAAACAC CGGCGGAGGG1561 GGTCCTGGGT CTCATGGACC TATCTCTAAC GGACATGTTA ACGGAAACAC CGGCGGAGGG
1621 TCGTCAAGAG TCGGTGGATC GCTTCTCACG GTAGCTTTTC AAATATTGGT AAACAGTTTA1621 TCGTCAAGAG TCGGTGGATC GCTTCTCACG GTAGCTTTTC AAATATTGGT AAACAGTTTA
1681 CCAACGGCTA AACTGACCGT CGAATCGGTT GAAACGGTTA ATAATC GAT TTCGTGTACT1681 CCAACGGCTA AACTGACCGT CGAATCGGTT GAAACGGTTA ATAATC GAT TTCGTGTACT
1741 GTCCAAAAGA TCAAAGCTGC CCTTTCAATG CGAAAGTTGA TGACTCAGTG AGTTGGGTAA1741 GTCCAAAAGA TCAAAGCTGC CCTTTCAATG CGAAAGTTGA TGACTCAGTG AGTTGGGTAA
18 01 TTTTAGTTAT TAATATAGGT TTTGGTTGTG GTTAAAGAGT GTGCGACTCA GTAAGTTGGG18 01 TTTTAGTTAT TAATATAGGT TTTGGTTGTG GTTAAAGAGT GTGCGACTCA GTAAGTTGGG
1861 ACGAGTGGGG GTATTGAGTC AAGAACGAAC CGCGCGTGGA GAATCATTGT ATGGTGGTGA1861 ACGAGTGGGG GTATTGAGTC AAGAACGAAC CGCGCGTGGA GAATCATTGT ATGGTGGTGA
1921 TCAAAGGGAC AAGGGTGGTG GTTCGGGTAT TGACTCAGGT CGACCCTTTG AGTTGGCTCG1921 TCAAAGGGAC AAGGGTGGTG GTTCGGGTAT TGACTCAGGT CGACCCTTTG AGTTGGCTCG
1981 CCGGGACCAG AAAAGAAAGT CAAAACTCAA ACAGCGTCGT ATTTGGTGTA CTAATCCAAT1981 CCGGGACCAG AAAAGAAAGT CAAAACTCAA ACAGCGTCGT ATTTGGTGTA CTAATCCAAT
2 041 TCTCTTTTC CTAGGGATTT AATGTCAATG AACATTATTG TATTGAGTTG TCACTTTCTT2 041 TCTCTTTTC CTAGGGATTT AATGTCAATG AACATTATTG TATTGAGTTG TCACTTTCTT
2101 TTTTTAGGGG TTTGGCTTTT GGAAACTTAC TAATTTAA A TATTAATTTC ACACTTCAAT2101 TTTTTAGGGG TTTGGCTTTT GGAAACTTAC TAATTTAA A TATTAATTTC ACACTTCAAT
2161 TTAAAAAAAA AAAAAAAAA 根据已经获得的 SEQ ID NO: 9序列， 设计如下三条特异性引物， 作为反转录引 物及 5'RACE的 3 '端特异性引物。 2161 TTAAAAAAAA AAAAAAAAA According to the sequence of SEQ ID NO: 9 that has been obtained, the following three specific primers are designed as reverse transcription primers. And 3'-end specific primers for 5' RACE.
GhHD-2 GSP 1： SEQ ID NO: 10： GhHD-2 GSP 1: SEQ ID NO: 10:
CAAGTTCATCCATGGCAGCC  CAAGTTCATCCATGGCAGCC
GhHD-2 GSP2: SEQ ID NO: 1 1：  GhHD-2 GSP2: SEQ ID NO: 1 1:
GATCGAGTGTTGTTGCCGGTC  GATCGAGTGTTGTTGCCGGTC
GhHD-2 GSP3 : SEQ ID NO: 12：  GhHD-2 GSP3 : SEQ ID NO: 12:
ACTAATTGTGGTGGTCCGGC  ACTAATTGTGGTGGTCCGGC
试剂盒自带通用引物：  The kit comes with universal primers:
AAP: SEQ ID NO: 13：  AAP: SEQ ID NO: 13:
GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG 实验步骤按试剂盒说明书操作（ 5 ' RACE System for Rapid Amplification of cDNA Ends 试剂盒购自 invitrogen公司）。  The GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
以 GhHD-2 GSP1 ( SEQ ID NO: 10) 为反转录引物， 以棉花 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: 10与通用引物 SEQ ID NO: 13 (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：  GhHD-2 GSP1 (SEQ ID NO: 10) was used as a reverse transcription primer, and reverse transcription was performed using cotton mRNA as a template to obtain a cDNA template, and then Poly C tail was added according to the procedure in the above 5' RACE kit instructions. The tailed product was subjected to the first round of PCR amplification using the primer. The primer used was SEQ ID NO: 10 and the universal primer SEQ ID NO: 13 (the kit is self-contained, and I is a hypoxanthine modified a, c, g). , or t), the specific steps are as follows:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ mRNA反转录 的 cDNA, 1.0 μΐ Ex Taq (购自 TAKARA)、 10 μΜ的引物 SEQ ID NO: 10和 SEQ ID NO: 13 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 30 s， 58°C退火 30 s， 72 °C 延伸 lmin)， 72 °C 延伸 10 min。  50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΐ Ex Taq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 10 and SEQ ID NO: 13 each of 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min), 72 °C for 10 min.
所得的 PCR产物用双蒸水稀释 50倍后取 2.0 μΐ作为模板，用 SEQ ID NO: 11与通用 引物 SEQ ID NO: 8进行第二轮 PCR扩增， 具体步骤如下：  The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 11 and the universal primer SEQ ID NO: 8. The specific steps are as follows:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ稀释的第一轮 PCR产物， 1.0 l Ex Taq、 10 μΜ的引物 SEQ ID NO: 11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 min， 33个循环 （ 94 变性 30 s， 53 °C 退火 30 s， 72V 延伸 2 min)， 72 °C 延伸 10 min。 回收第二次 PCR产物中约为 900 bp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 μ§/η 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%， -80°C保存备用。 用引物 SEQ ID NO: 11与 3'端引物 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 （GhHD21-l， GhHD21-3 ; GhHD22-5 ) ，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 GhHD22-5测序获得序列为 SEQ ID NO: 14：50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 l Ex Taq, 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 8 2.0 μl, and 35 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (94 denaturation for 30 s, annealing at 53 °C for 30 s, 72V extension for 2 min), extension at 72 °C for 10 min. A strip of approximately 900 bp in the second PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy Vector, then converted to JM109 (specifically the same as above), randomly picked 10 White colonies were inoculated separately in LB liquid medium containing 50 μ § /η ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. PCR amplification of the bacterial solution using the primers SEQ ID NO: 11 and the 3' primer SEQ ID NO: 12 (reaction system and reaction) The conditions were the same as above. Three positive clones (GhHD21-l, GhHD21-3 ; GhHD22-5) were obtained and sequenced and sequenced by Yingjiejieji (Shanghai) Trading Co., Ltd. to obtain a 5' end sequence of the cDNA of the gene. The resulting 5' RACE product clone GhHD22-5 was sequenced to obtain the sequence of SEQ ID NO: 14:
1 T GA GAATT TTGGGGGGTT TATAGATGAT AGTTCTGGTC CTAATGACGG TCTTGGTGGT1 T GA GAATT TTGGGGGGTT TATAGATGAT AGTTCTGGTC CTAATGACGG TCTTGGTGGT
61 GCAAGAA AG TTGCAGACGT ACCTTATAAC ACCCCCACCA TGCCTACTGG TGTTTTCTCT61 GCAAGAA AG TTGCAGACGT ACCTTATAAC ACCCCCACCA TGCCTACTGG TGTTTTCTCT
121 CAACCCCGCC TTGTTTCTTC TTCAATTCCT AAGAACATGT TCAACTCACC AGGCCTTTCT121 CAACCCCGCC TTGTTTCTTC TTCAATTCCT AAGAACATGT TCAACTCACC AGGCCTTTCT
181 CTTGCTCTTC AACCCAATAT AGA AATCAA GGAGATGAGA CTAGATTAGG TGAGAATTTT181 CTTGCTCTTC AACCCAATAT AGA AATCAA GGAGATGAGA CTAGATTAGG TGAGAATTTT
241 GAAGGAAGTA TTGGGAGAAG AAGCAGAGAA GAAGAACATG AAAGTAGATC TGGGAGTGAT241 GAAGGAAGTA TTGGGAGAAG AAGCAGAGAA GAAGAACATG AAAGTAGATC TGGGAGTGAT
3 01 AATATGGATG GTGGTTCCGG TGATGATCAT GACCCCACCA CCGCGGCAGG TGATAAACCG3 01 AATATGGATG GTGGTTCCGG TGATGATCAT GACCCCACCA CCGCGGCAGG TGATAAACCG
361 CCGAGGAAAA AGAGA ACCA CCGTCATACA CCTCAACAAA TCCAAGAGCT TGAAGCTCTC361 CCGAGGAAAA AGAGA ACCA CCGTCATACA CCTCAACAAA TCCAAGAGCT TGAAGCTCTC
421 TTTAAGGAGT GTCCTCATCC AGA GAAAAA CAGAGATTAG AGCTTAGTAA AAGGCTTTGT421 TTTAAGGAGT GTCCTCATCC AGA GAAAAA CAGAGATTAG AGCTTAGTAA AAGGCTTTGT
481 TTAGAAACCA GACAAGTTAA GTTTTGGTTC CAAAACAGGC GTACTCAAAT GAAGACACAA481 TTAGAAACCA GACAAGTTAA GTTTTGGTTC CAAAACAGGC GTACTCAAAT GAAGACACAA
541 TTAGAGCGAC ATGAGAACTC ATTGTTGAGA CAGGAGAATG ATAAGCTTAG AGCTGAAAAC541 TTAGAGCGAC ATGAGAACTC ATTGTTGAGA CAGGAGAATG ATAAGCTTAG AGCTGAAAAC
601 ATGTCTATAA GAGATGCAAT GAGGAATCCT ATATGTACTA ATTGTGGTGG TCCGGCTATT601 ATGTCTATAA GAGATGCAAT GAGGAATCCT ATATGTACTA ATTGTGGTGG TCCGGCTATT
661 ATTGGTGATA TGTCACTTGA AGAACAACTT CTTAGAATCG AAAATGCTCG TTTAAAAGAT661 ATTGGTGATA TGTCACTTGA AGAACAACTT CTTAGAATCG AAAATGCTCG TTTAAAAGAT
721 GAAT AGA C GTGTTTGTGC ACTTGCTGGT AAGTTTTTAG GTCGTCCAAT TACAGGACCT721 GAAT AGA C GTGTTTGTGC ACTTGCTGGT AAGTTTTTAG GTCGTCCAAT TACAGGACCT
781 CCATTACCAA ACTCAAGTTT AGAGCTTGGT GTTGGCACCA ATGGTACTTT TGGAACCACT781 CCATTACCAA ACTCAAGTTT AGAGCTTGGT GTTGGCACCA ATGGTACTTT TGGAACCACT
841 ATGGCTACTA CTACAACATT GCCTTTAGGA CATGATGCTT ACCAACAAT GGTTGTTCCT841 ATGGCTACTA CTACAACATT GCCTTTAGGA CATGATGCTT ACCAACAAT GGTTGTTCCT
901 AGTAATAGAC CGGCAACAAC ACTCGATCGA C 901 AGTAATAGAC CGGCAACAAC ACTCGATCGA C
将 5'RACE获得的序列 SEQ ID NO: 14， 与 3'RACE获得的序列 SEQ ID NO: 9拼 接， 获得 SEQ ID NO: 15:  The sequence obtained by 5' RACE, SEQ ID NO: 14, was ligated with the sequence obtained by 3' RACE, SEQ ID NO: 9, to obtain SEQ ID NO: 15:
1 TTGATGAATT TTGGGGGGTT TATAGATGAT AGTTCTGGTC CTAATGACGG TCTTGGTGGT 1 TTGATGAATT TTGGGGGGTT TATAGATGAT AGTTCTGGTC CTAATGACGG TCTTGGTGGT
61 GCAAGAATAG TTGCAGACGT ACCTTATAAC ACCCCCACCA TGCCTACTGG TGTTTTCTCT61 GCAAGAATAG TTGCAGACGT ACCTTATAAC ACCCCCACCA TGCCTACTGG TGTTTTCTCT
121 CAACCCCGCC TTGTTTCTTC TTCAATTCCT AAGAACATGT TCAACTCACC AGGCCTTTCT121 CAACCCCGCC TTGTTTCTTC TTCAATTCCT AAGAACATGT TCAACTCACC AGGCCTTTCT
181 CTTGCTCTTC AACCCAATAT AGATAATCAA GGAGATGAGA CTAGATTAGG TGAGAATTTT181 CTTGCTCTTC AACCCAATAT AGATAATCAA GGAGATGAGA CTAGATTAGG TGAGAATTTT
241 GAAGGAAGTA TTGGGAGAAG AAGCAGAGAA GAAGAACATG AAAGTAGATC TGGGAGTGAT241 GAAGGAAGTA TTGGGAGAAG AAGCAGAGAA GAAGAACATG AAAGTAGATC TGGGAGTGAT
3 01 AATATGGATG GTGGTTCCGG TGATGATCAT GACCCCACCA CCGCGGCAGG TGATAAACCG3 01 AATATGGATG GTGGTTCCGG TGATGATCAT GACCCCACCA CCGCGGCAGG TGATAAACCG
361 CCGAGGAAAA AGAGATACCA CCGTCATACA CCTCAACAAA TCCAAGAGCT TGAAGCTCTC361 CCGAGGAAAA AGAGATACCA CCGTCATACA CCTCAACAAA TCCAAGAGCT TGAAGCTCTC
421 TTTAAGGAGT GTCCTCATCC AGATGAAAAA CAGAGATTAG AGCTTAGTAA AAGGCTTTGT421 TTTAAGGAGT GTCCTCATCC AGATGAAAAA CAGAGATTAG AGCTTAGTAA AAGGCTTTGT
481 TTAGAAACCA GACAAGTTAA GTTTTGGTTC CAAAACAGGC GTACTCAAAT GAAGACACAA481 TTAGAAACCA GACAAGTTAA GTTTTGGTTC CAAAACAGGC GTACTCAAAT GAAGACACAA
541 TTAGAGCGAC ATGAGAACTC ATTGTTGAGA CAGGAGAATG ATAAGCTTAG AGCTGAAAAC541 TTAGAGCGAC ATGAGAACTC ATTGTTGAGA CAGGAGAATG ATAAGCTTAG AGCTGAAAAC
601 ATGTCTATAA GAGATGCAAT GAGGAATCCT ATATGTACTA ATTGTGGTGG TCCGGCTATT601 ATGTCTATAA GAGATGCAAT GAGGAATCCT ATATGTACTA ATTGTGGTGG TCCGGCTATT
661 ATTGGTGATA TGTCACTTGA AGAACAACTT CTTAGAATCG AAAATGCTCG TTTAAAAGAT661 ATTGGTGATA TGTCACTTGA AGAACAACTT CTTAGAATCG AAAATGCTCG TTTAAAAGAT
721 GAATTAGATC GTGTTTGTGC ACTTGCTGGT AAGTTTTTAG GTCGTCCAAT TACAGGACCT721 GAATTAGATC GTGTTTGTGC ACTTGCTGGT AAGTTTTTAG GTCGTCCAAT TACAGGACCT
781 CCATTACCAA ACTCAAGTTT AGAGCTTGGT GTTGGCACCA ATGGTACTTT TGGAACCACT781 CCATTACCAA ACTCAAGTTT AGAGCTTGGT GTTGGCACCA ATGGTACTTT TGGAACCACT
841 ATGGCTACTA CTACAACATT GCCTTTAGGA CATGATGCTT TACCAACAAT GGTTGTTCCT841 ATGGCTACTA CTACAACATT GCCTTTAGGA CATGATGCTT TACCAACAAT GGTTGTTCCT
901 AGTAATAGAC CGGCAACAAC ACTCGATCGA TCGATGTTTT TGGAACTTGC TTTGGCTGCC901 AGTAATAGAC CGGCAACAAC ACTCGATCGA TCGATGTTTT TGGAACTTGC TTTGGCTGCC
961 ATGGATGAAC TTGTTAAGAT GGCACAAACT GATGAGCCAT TATGGATTAA GAACATAGAA961 ATGGATGAAC TTGTTAAGAT GGCACAAACT GATGAGCCAT TATGGATTAA GAACATAGAA
1021 GGTGGAAGAG AAATGTTGAA CCATGATGAG TATTTAAGGA CATTTACACC TTGTATTGGT 1081 TTAAAACCAA ATGGTTTTGT CACTGAAGCG TCAAGGGAGA CTGGTGTAGT GATTATCAAC1021 GGTGGAAGAG AAATGTTGAA CCATGATGAG TATTTAAGGA CATTTACACC TTGTATTGGT 1081 TTAAAACCAA ATGGTTTTGT CACTGAAGCG TCAAGGGAGA CTGGTGTAGT GATTATCAAC
1141 AGTCTAGCCC TTGTTGAAAC ATTAATGGAC TCGAATCGTT GGGCAGAGAT GTTTCATTGT1141 AGTCTAGCCC TTGTTGAAAC ATTAATGGAC TCGAATCGTT GGGCAGAGAT GTTTCATTGT
12 01 ATGATTGCTA GAACA CAAC AACTGATGTG ATATCTAATG GCATGGGAGG AACCAGAAAT12 01 ATGATTGCTA GAACA CAAC AACTGATGTG ATATCTAATG GCATGGGAGG AACCAGAAAT
1261 GGTGCACTTC AACTTATGAA TGCTGAGCTT CAAATCCTTT CACCTTTAGT TCCTGTTCGT1261 GGTGCACTTC AACTTATGAA TGCTGAGCTT CAAATCCTTT CACCTTTAGT TCCTGTTCGT
1321 GAAGTAAGTT TCTTAAGGTT CTGTAAACAA CATGCTGAAG GTGTTTGGGC TGTGGTTGAT1321 GAAGTAAGTT TCTTAAGGTT CTGTAAACAA CATGCTGAAG GTGTTTGGGC TGTGGTTGAT
1381 GTATCCGTTG ATACTATCAA AGAAAGTACT ACATTTGTTA CCTGTAGGAG ACTTCCTTCT1381 GTATCCGTTG ATACTATCAA AGAAAGTACT ACATTTGTTA CCTGTAGGAG ACTTCCTTCT
1441 GGTTGTGTTG TTCAAGA AT GCCTAATGGT TACTCCAAGG TTATATGGGT TGAACATGCT1441 GGTTGTGTTG TTCAAGA AT GCCTAATGGT TACTCCAAGG TTATATGGGT TGAACATGCT
1501 GAA A GA G AGAGCCAAGT TCATCAACTA TACCGGCCTT TACTAAGTTC CGGCGTGGGC1501 GAA A GA G AGAGCCAAGT TCATCAACTA TACCGGCCTT TACTAAGTTC CGGCGTGGGC
1561 TTCGGTGCCC AACGGTGGGT GGCGGCCCTT CAACGGCAAT GCGAATGCCT TGCCATACTC1561 TTCGGTGCCC AACGGTGGGT GGCGGCCCTT CAACGGCAAT GCGAATGCCT TGCCATACTC
1621 ATGTCCTCCA CCGTTCCCAC TAGAGACCAC ACCGCTATAA CTGCGAGTGG GAGACGGAGC1621 ATGTCCTCCA CCGTTCCCAC TAGAGACCAC ACCGCTATAA CTGCGAGTGG GAGACGGAGC
1681 ATGTTAAAGC TAGCTCAACG GATGACAGAT AACTTCTGTG CCGGGGTTTG TGCATCGACA1681 ATGTTAAAGC TAGCTCAACG GATGACAGAT AACTTCTGTG CCGGGGTTTG TGCATCGACA
1741 GTTCATAAAT GGAACAAGCT TAACGCCGGG AATGTAGACG AAGACGTTAG GGTTATGACT1741 GTTCATAAAT GGAACAAGCT TAACGCCGGG AATGTAGACG AAGACGTTAG GGTTATGACT
18 01 AGGAAAAGCA TTGACGACCC CGGCGAACCG CCAGGGATCG TACTAAGCGC CGCCACTTCG18 01 AGGAAAAGCA TTGACGACCC CGGCGAACCG CCAGGGATCG TACTAAGCGC CGCCACTTCG
1861 GTTTGGTTGC CGGTGTCACC GCAACGGCTT TTCGATTTCC TACGCGACGA ACGGTTGAGG1861 GTTTGGTTGC CGGTGTCACC GCAACGGCTT TTCGATTTCC TACGCGACGA ACGGTTGAGG
1921 AGCGAGTGGG ACATATTGTC AAATGGCGGA CCTATGCAAG AGATGGCACA CATTGCCAAA1921 AGCGAGTGGG ACATATTGTC AAATGGCGGA CCTATGCAAG AGATGGCACA CATTGCCAAA
1981 GGCCAAGATC ATGGCAATTG CGTTTCCCTC CTGCGTGCCA GTGCCATGAA CGCAAATCAG1981 GGCCAAGATC ATGGCAATTG CGTTTCCCTC CTGCGTGCCA GTGCCATGAA CGCAAATCAG
2 041 AGCAGCATGC TGATATTGCA GGAAACATGC ATAGACGCAG CAGGGTCGCT TGTAGTGTAC2 041 AGCAGCATGC TGATATTGCA GGAAACATGC ATAGACGCAG CAGGGTCGCT TGTAGTGTAC
2101 GCGCCAGTTG ATATTCCAGC CATGCACGTT GTCATGAACG GTGGTGATTC CGCTTACGTC2101 GCGCCAGTTG ATATTCCAGC CATGCACGTT GTCATGAACG GTGGTGATTC CGCTTACGTC
2161 GCACTTTTAC CTTCGGGATT CGCCATTGTC CCGGACGGTC CTGGGTCTCA TGGACCTATC2161 GCACTTTTAC CTTCGGGATT CGCCATTGTC CCGGACGGTC CTGGGTCTCA TGGACCTATC
2221 TCTAACGGAC ATGTTAACGG AAACACCGGC GGAGGGTCGT CAAGAGTCGG TGGATCGCTT2221 TCTAACGGAC ATGTTAACGG AAACACCGGC GGAGGGTCGT CAAGAGTCGG TGGATCGCTT
2281 CTCACGGTAG CTTTTCAAAT ATTGGTAAAC AGTTTACCAA CGGCTAAACT GACCGTCGAA2281 CTCACGGTAG CTTTTCAAAT ATTGGTAAAC AGTTTACCAA CGGCTAAACT GACCGTCGAA
2341 TCGGTTGAAA CGGTTAATAA TCTGATTTCG TGTACTGTCC AAAAGA CAA AGCTGCCCTT2341 TCGGTTGAAA CGGTTAATAA TCTGATTTCG TGTACTGTCC AAAAGA CAA AGCTGCCCTT
24 01 TCAATGCGAA AGTTGATGAC TCAGTGAGTT GGGTAATTTT AGTTATTAAT ATAGGTTTTG24 01 TCAATGCGAA AGTTGATGAC TCAGTGAGTT GGGTAATTTT AGTTATTAAT ATAGGTTTTG
2461 GTTGTGGTTA AAGAGTGTGC GACTCAGTAA GTTGGGACGA GTGGGGGTAT TGAGTCAAGA2461 GTTGTGGTTA AAGAGTGTGC GACTCAGTAA GTTGGGACGA GTGGGGGTAT TGAGTCAAGA
2521 ACGAACCGCG CGTGGAGAAT CATTGTATGG TGGTGATCAA AGGGACAAGG GTGGTGGTTC2521 ACGAACCGCG CGTGGAGAAT CATTGTATGG TGGTGATCAA AGGGACAAGG GTGGTGGTTC
2581 GGGTATTGAC TCAGGTCGAC CCTTTGAGTT GGCTCGCCGG GACCAGAAAA GAAAGTCAAA2581 GGGTATTGAC TCAGGTCGAC CCTTTGAGTT GGCTCGCCGG GACCAGAAAA GAAAGTCAAA
2641 ACTCAAACAG CGTCGTATTT GGTGTACTAA TCCAATTTCT CTTTTCCTAG GGATTTAATG2641 ACTCAAACAG CGTCGTATTT GGTGTACTAA TCCAATTTCT CTTTTCCTAG GGATTTAATG
2701 TCAATGAACA TTATTGTATT GAGTTGTCAC TTCTTTTTT TAGGGGTTTG GCTTTTGGAA2701 TCAATGAACA TTATTGTATT GAGTTGTCAC TTCTTTTTT TAGGGGTTTG GCTTTTGGAA
2761 ACTTACTAAT TTAATATATT AATTTCACAC TTCAATTTAA AAAAAAAAAA AAAAA 根据 SEQ ID NO: 15序列设计一对引物如下: 2761 ACTTACTAAT TTAATATATT AATTTCACAC TTCAATTTAA AAAAAAAAAA AAAAA A pair of primers were designed according to the sequence of SEQ ID NO: 15 as follows:
GhHDbZIP-lF: SEQ ID NO: 16： GhHDbZIP-lF: SEQ ID NO: 16:
TTGATGAATTTTGGGGGGTT  TTGATGAATTTTGGGGGGTT
GhHDbZIP-lR: SEQ ID NO: 17：  GhHDbZIP-lR: SEQ ID NO: 17:
CATCAACTTTCGCATTGAAAG 通过 SEQ ID NO: 16和 SEQ ID NO: 17来克隆 GhHDbZIP-1全长编码序列。  CATCAACTTTCGCATTGAAAG The GhHDbZIP-1 full-length coding sequence was cloned by SEQ ID NO: 16 and SEQ ID NO: 17.
采用 stratagene的 PfUUltra II Fusion HS DNA Polymerase, 以棉花的 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: 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 2min， 35个循环（95°C 变性 25 s， 54°C退火 30 s， 72 °C 延伸 lmin30s)， 72 °C 延伸 5 min。 PCR was carried out using strafgene's PfUUltra II Fusion HS DNA Polymerase using cotton cDNA as a template. 50 μΐ PCR reaction system: 5 μΐ lO PfuUltra II reaction Buffer, 0.5 μΐ 25 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ primers SEQ ID NO: 20 and SEQ ID NO: 21 each 2.0 μl, and 37.5 μM 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 54 °C for 30 s, extension at 72 °C for 1 min 30 s), extension at 72 °C for 5 min.
PCR扩增产物加 A尾： PCR产物加 2.5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 ΙΟ Εχ Buffer, 0.5 μΐ 5 mM的 dATP ， 1.0 μΐ Ex Taq。反应条件： 70°C反应 30分钟。将得到约 2.4 Kbp的 DNA片段回收（Omega 回收试剂盒） ， 连接至 pGEM T-easy载体上 （得到 GhHDbZIP-1-pGEM质粒） ，然后 转化 JM109(方法同上）， 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终浓度 20%， -80°C保存备用。 用引物 SEQ ID NO: 16与 SEQ ID NO: 17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 SEQIDNO: 2， 其编码的蛋白的氨基酸序列为 SEQIDNO: 1  The PCR amplification product was added with A tail: The PCR product was added 2.5 times of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, and the supernatant was removed, dried, and dissolved in 21 μl of double distilled water. Add 2.5 μΐ Εχ Εχ Buffer, 0.5 μΐ 5 mM dATP, 1.0 μΐ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 2.4 Kbp was recovered (Omega recovery kit), ligated into the pGEM T-easy vector (GhHDbZIP-1-pGEM plasmid was obtained), and then transformed into JM109 (method as above), and 8 white colonies were randomly picked. The cells were inoculated in an LB liquid medium containing 50 g/mL ampicillin, cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20%, and stored at -80 ° C until use. The primers SEQ ID NO: 16 and SEQ ID NO: 17 were used for PCR amplification (reaction system and reaction conditions are the same as above), and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. SEQ ID NO: 2, the amino acid sequence of the encoded protein is SEQ ID NO: 1
HDbZIP-1蛋白的氨基酸序列： SEQ ID NO: 1 Amino acid sequence of HDbZIP-1 protein: SEQ ID NO: 1
1 NFGGFIDDS SGPNDGLGGA RIVADVPYNT PT PTGVFSQ PRLVSSSIPK N FNSPGLSL 1 NFGGFIDDS SGPNDGLGGA RIVADVPYNT PT PTGVFSQ PRLVSSSIPK N FNSPGLSL
61 ALQPNIDNQG DETRLGENFE GSIGRRSREE EHESRSGSDN DGGSGDDHD PTTAAGDKPP61 ALQPNIDNQG DETRLGENFE GSIGRRSREE EHESRSGSDN DGGSGDDHD PTTAAGDKPP
121 RKKRYHRHTP QQIQELEALF KECPHPDEKQ RLELSKRLCL ETRQVKFWFQ NRRTQ KTQL121 RKKRYHRHTP QQIQELEALF KECPHPDEKQ RLELSKRLCL ETRQVKFWFQ NRRTQ KTQL
181 ERHENSLLRQ ENDKLRAEN SIRDA RNPI CTNCGGPAII GD SLEEQLL RIENARLKDE181 ERHENSLLRQ ENDKLRAEN SIRDA RNPI CTNCGGPAII GD SLEEQLL RIENARLKDE
241 LDRVCALAGK FLGRPITGPP LPNSSLELGV GTNGTFGTT ATTTTLPLGH DALPT VVPS241 LDRVCALAGK FLGRPITGPP LPNSSLELGV GTNGTFGTT ATTTTLPLGH DALPT VVPS
301 NRPATTLDRS FLELALAA DELVK AQTD EPLWIKNIEG GRE LNHDEY LRTFTPCIGL301 NRPATTLDRS FLELALAA DELVK AQTD EPLWIKNIEG GRE LNHDEY LRTFTPCIGL
361 KPNGFVTEAS RETGVVIINS LALVETL DS NRWAE FHC IARTSTTDVI SNG GGTRNG361 KPNGFVTEAS RETGVVIINS LALVETL DS NRWAE FHC IARTSTTDVI SNG GGTRNG
421 ALQL NAELQ ILSPLVPVRE VSFLRFCKQH AEGVWAVVDV SVDTIKESTT FVTCRRLPSG421 ALQL NAELQ ILSPLVPVRE VSFLRFCKQH AEGVWAVVDV SVDTIKESTT FVTCRRLPSG
481 CVVQD PNGY SKVIWVEHAE YDESQVHQLY RPLLSSGVGF GAQRWVAALQ RQCECLAIL481 CVVQD PNGY SKVIWVEHAE YDESQVHQLY RPLLSSGVGF GAQRWVAALQ RQCECLAIL
541 SSTVPTRDHT AITASGRRS LKLAQR TDN FCAGVCASTV HKWNKLNAGN VDEDVRV TR541 SSTVPTRDHT AITASGRRS LKLAQR TDN FCAGVCASTV HKWNKLNAGN VDEDVRV TR
601 KSIDDPGEPP GIVLSAATSV WLPVSPQRLF DFLRDERLRS EWDILSNGGP QE AHIAKG601 KSIDDPGEPP GIVLSAATSV WLPVSPQRLF DFLRDERLRS EWDILSNGGP QE AHIAKG
661 QDHGNCVSLL RASA NANQS S LILQETCI DAAGSLVVYA PVDIPA HVV NGGDSAYVA661 QDHGNCVSLL RASA NANQS S LILQETCI DAAGSLVVYA PVDIPA HVV NGGDSAYVA
721 LLPSGFAIVP DGPRSHGPIS NGHVNGNTGG GSSSVGGSLL TVAFQILVNS LPTAKLTVES721 LLPSGFAIVP DGPRSHGPIS NGHVNGNTGG GSSSVGGSLL TVAFQILVNS LPTAKLTVES
781 VETVNNLISC TVQKIKAALQ CES* 781 VETVNNLISC TVQKIKAALQ CES*
GhHDbZIP-1编码基因的核苷酸序列： SEQIDNO: 2 Nucleotide sequence of the GhHDbZIP-1 encoding gene: SEQ ID NO: 2
1 ATGAATTTTG GGGGGTTTAT AGATGATAGT TCTGGTCCTA ATGACGGTCT TGGTGGTGCA 1 ATGAATTTTG GGGGGTTTAT AGATGATAGT TCTGGTCCTA ATGACGGTCT TGGTGGTGCA
61 AGAATAGTTG CAGACGTACC TTATAACACC CCCACCATGC CTACTGGTGT TTTCTCTCAA61 AGAATAGTTG CAGACGTACC TTATAACACC CCCACCATGC CTACTGGTGT TTTCTCTCAA
121 CCCCGCCTTG TTCTTCTTC AATTCCTAAG AACATGTTCA ACTCACCAGG CCTTTCTCTT121 CCCCGCCTTG TTCTTCTTC AATTCCTAAG AACATGTTCA ACTCACCAGG CCTTTCTCTT
181 GCTCTTCAAC CCAA A AGA TAATCAAGGA GATGAGACTA GATTAGGTGA GAATTTTGAA 241 GGAAGTATTG GGAGAAGAAG CAGAGAAGAA GAACATGAAA GTAGATCTGG GAGTGATAAT181 GCTCTTCAAC CCAA A AGA TAATCAAGGA GATGAGACTA GATTAGGTGA GAATTTTGAA 241 GGAAGTATTG GGAGAAGAAG CAGAGAAGAA GAACATGAAA GTAGATCTGG GAGTGATAAT
3 01 ATGGATGGTG GTTCCGGTGA TGATCATGAC CCCACCACCG CGGCAGGTGA TAAACCGCCG3 01 ATGGATGGTG GTTCCGGTGA TGATCATGAC CCCACCACCG CGGCAGGTGA TAAACCGCCG
361 AGGAAAAAGA GATACCACCG TCATACACCT CAACAAATCC AAGAGCTTGA AGCTCTCTTT361 AGGAAAAAGA GATACCACCG TCATACACCT CAACAAATCC AAGAGCTTGA AGCTCTCTTT
421 AAGGAGTGTC CTCATCCAGA TGAAAAACAG AGATTAGAGC TTAGTAAAAG GCTTTGTTTA421 AAGGAGTGTC CTCATCCAGA TGAAAAACAG AGATTAGAGC TTAGTAAAAG GCTTTGTTTA
481 GAAACCAGAC AAGTTAAGTT TTGGTTCCAA AACAGGCGTA CTCAAATGAA GACACAATTA481 GAAACCAGAC AAGTTAAGTT TTGGTTCCAA AACAGGCGTA CTCAAATGAA GACACAATTA
541 GAGCGACATG AGAACTCATT GTTGAGACAG GAGAA GA A AGCTTAGAGC TGAAAACATG541 GAGCGACATG AGAACTCATT GTTGAGACAG GAGAA GA A AGCTTAGAGC TGAAAACATG
601 TCTATAAGAG ATGCAATGAG GAATCC A A TGTACTAATT GTGGTGGTCC GGCTATTATT601 TCTATAAGAG ATGCAATGAG GAATCC A A TGTACTAATT GTGGTGGTCC GGCTATTATT
661 GGTGATATGT CACTTGAAGA ACAACTTCTT AGAATCGAAA ATGCTCGTTT AAAAGA GAA661 GGTGATATGT CACTTGAAGA ACAACTTCTT AGAATCGAAA ATGCTCGTTT AAAAGA GAA
721 TTAGATCGTG TTTGTGCACT TGCTGGTAAG TTTTTAGGTC GTCCAATTAC AGGACCTCCA721 TTAGATCGTG TTTGTGCACT TGCTGGTAAG TTTTTAGGTC GTCCAATTAC AGGACCTCCA
781 TTACCAAACT CAAGTTTAGA GCTTGGTGTT GGCACCAATG GTACTTTTGG AACCACTATG781 TTACCAAACT CAAGTTTAGA GCTTGGTGTT GGCACCAATG GTACTTTTGG AACCACTATG
841 GCTACTACTA CAACATTGCC TTTAGGACAT GATGCTTTAC CAACAATGGT TGTTCCTAGT841 GCTACTACTA CAACATTGCC TTTAGGACAT GATGCTTTAC CAACAATGGT TGTTCCTAGT
901 AATAGACCGG CAACAACACT CGATCGATCG ATGTTTTTGG AACTTGCTTT GGCTGCCATG901 AATAGACCGG CAACAACACT CGATCGATCG ATGTTTTTGG AACTTGCTTT GGCTGCCATG
961 GATGAACTTG TTAAGATGGC ACAAACTGAT GAGCCATTAT GGATTAAGAA CATAGAAGGT961 GATGAACTTG TTAAGATGGC ACAAACTGAT GAGCCATTAT GGATTAAGAA CATAGAAGGT
1021 GGAAGAGAAA TGTTGAACCA TGATGAGTAT TTAAGGACAT TTACACCTTG TATTGGTTTA1021 GGAAGAGAAA TGTTGAACCA TGATGAGTAT TTAAGGACAT TTACACCTTG TATTGGTTTA
1081 AAACCAAATG GTTTTGTCAC TGAAGCGTCA AGGGAGACTG GTGTAGTGAT TA CAACAGT1081 AAACCAAATG GTTTTGTCAC TGAAGCGTCA AGGGAGACTG GTGTAGTGAT TA CAACAGT
1141 CTAGCCCTTG TTGAAACATT AATGGACTCG AATCGTTGGG CAGAGATGTT TCATTGTATG1141 CTAGCCCTTG TTGAAACATT AATGGACTCG AATCGTTGGG CAGAGATGTT TCATTGTATG
12 01 ATTGC AGAA CATCAACAAC TGATGTGATA TCTAATGGCA TGGGAGGAAC CAGAAATGGT12 01 ATTGC AGAA CATCAACAAC TGATGTGATA TCTAATGGCA TGGGAGGAAC CAGAAATGGT
1261 GCACTTCAAC A GAATGC TGAGCTTCAA ATCCTTTCAC CTTTAGTTCC TGTTCGTGAA1261 GCACTTCAAC A GAATGC TGAGCTTCAA ATCCTTTCAC CTTTAGTTCC TGTTCGTGAA
1321 GTAAGTTTCT TAAGGTTCTG TAAACAACAT GCTGAAGGTG TTTGGGCTGT GGTTGATGTA1321 GTAAGTTTCT TAAGGTTCTG TAAACAACAT GCTGAAGGTG TTTGGGCTGT GGTTGATGTA
1381 TCCGTTGATA C A CAAAGA AAGTACTACA TTTGTTACCT GTAGGAGACT TCCTTCTGGT1381 TCCGTTGATA C A CAAAGA AAGTACTACA TTTGTTACCT GTAGGAGACT TCCTTCTGGT
1441 TGTGTTGTTC AAGATATGCC TAATGGTTAC TCCAAGGTTA TATGGGTTGA ACATGCTGAA1441 TGTGTTGTTC AAGATATGCC TAATGGTTAC TCCAAGGTTA TATGGGTTGA ACATGCTGAA
1501 TATGATGAGA GCCAAGTTCA TCAACTATAC CGGCCTTTAC TAAGTTCCGG CGTGGGCTTC1501 TATGATGAGA GCCAAGTTCA TCAACTATAC CGGCCTTTAC TAAGTTCCGG CGTGGGCTTC
1561 GGTGCCCAAC GGTGGGTGGC GGCCCTTCAA CGGCAATGCG AATGCCTTGC CATACTCATG1561 GGTGCCCAAC GGTGGGTGGC GGCCCTTCAA CGGCAATGCG AATGCCTTGC CATACTCATG
1621 TCCTCCACCG TTCCCACTAG AGACCACACC GCTATAACTG CGAGTGGGAG ACGGAGCATG1621 TCCTCCACCG TTCCCACTAG AGACCACACC GCTATAACTG CGAGTGGGAG ACGGAGCATG
1681 TTAAAGC AG CTCAACGGAT GACAGATAAC TTCTGTGCCG GGGTTTGTGC ATCGACAGTT1681 TTAAAGC AG CTCAACGGAT GACAGATAAC TTCTGTGCCG GGGTTTGTGC ATCGACAGTT
1741 CATAAATGGA ACAAGCTTAA CGCCGGGAAT GTAGACGAAG ACGTTAGGGT TATGACTAGG1741 CATAAATGGA ACAAGCTTAA CGCCGGGAAT GTAGACGAAG ACGTTAGGGT TATGACTAGG
18 01 AAAAGCA G ACGACCCCGG CGAACCGCCA GGGATCGTAC TAAGCGCCGC CACTTCGGTT18 01 AAAAGCA G ACGACCCCGG CGAACCGCCA GGGATCGTAC TAAGCGCCGC CACTTCGGTT
1861 TGGTTGCCGG TGTCACCGCA ACGGCTTTTC GATTTCCTAC GCGACGAACG GTTGAGGAGC1861 TGGTTGCCGG TGTCACCGCA ACGGCTTTTC GATTTCCTAC GCGACGAACG GTTGAGGAGC
1921 GAGTGGGACA TATTGTCAAA TGGCGGACCT A GCAAGAGA TGGCACACAT TGCCAAAGGC1921 GAGTGGGACA TATTGTCAAA TGGCGGACCT A GCAAGAGA TGGCACACAT TGCCAAAGGC
1981 CAAGATCATG GCAATTGCGT TTCCCTCCTG CGTGCCAGTG CCATGAACGC AAATCAGAGC1981 CAAGATCATG GCAATTGCGT TTCCCTCCTG CGTGCCAGTG CCATGAACGC AAATCAGAGC
2 041 AGCATGTTGA TATTGCAGGA AACATGCATA GACGCAGCAG GGTCGCTTGT AGTGTACGCG2 041 AGCATGTTGA TATTGCAGGA AACATGCATA GACGCAGCAG GGTCGCTTGT AGTGTACGCG
2101 CCAGTTGATA TTCCAGCCAT GCACGTTGTC ATGAACGGTG GTGATTCCGC TTACGTCGCA2101 CCAGTTGATA TTCCAGCCAT GCACGTTGTC ATGAACGGTG GTGATTCCGC TTACGTCGCA
2161 CTTTTACCTT CGGGATTCGC CATTGTCCCG GACGGTCCTA GGTCTCATGG ACCTATCTCT2161 CTTTTACCTT CGGGATTCGC CATTGTCCCG GACGGTCCTA GGTCTCATGG ACCTATCTCT
2221 AACGGACATG TTAACGGAAA CACCGGCGGA GGGTCGTCGA GTGTCGGTGG ATCGCTTCTC2221 AACGGACATG TTAACGGAAA CACCGGCGGA GGGTCGTCGA GTGTCGGTGG ATCGCTTCTC
2281 ACGGTAGCTT TTCAAA A GGTAAACAGT TTACCAACGG CTAAACTGAC CGTCGAATCG2281 ACGGTAGCTT TTCAAA A GGTAAACAGT TTACCAACGG CTAAACTGAC CGTCGAATCG
2341 GTTGAAACGG TTAATAATCT GATTTCGTGT ACTGTCCAAA AGATCAAAGC TGCCCTTCAA2341 GTTGAAACGG TTAATAATCT GATTTCGTGT ACTGTCCAAA AGATCAAAGC TGCCCTTCAA
24 01 TGCGAAAGTT GA 实施例 3 GhHDbZIP-1基因植物表达载体构建 24 01 TGCGAAAGTT GA Example 3 Construction of GhHDbZIP-1 Gene Plant Expression Vector
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 GhHDbZIP-1基因的启动子和终止子。 The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as the plant expression vector, and the 35S promoter containing the double enhancer of the ΝΡΤΠ 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 GhHDbZIP-1 gene, respectively.
用引物 SEQ ID NO: 18和 SEQ ID NO: 19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: 18禾 Ρ 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 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 : 18 ：  Primer SEQ ID NO: 18 and SEQ ID NO: 19 were used to amplify Pnos with the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μl ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ ρΒΙ121, 1.0 μΐ PrimeSTAR, 10 μΜ primer SEQ ID 18: 18 and SEQ ID NO: 19 each 2.0 μl, and 31 μΐ 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 56 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min. After digestion with EcoRI and Bglll, the resulting PCR product was ligated into pCAMBIA2300 (promega, T4 ligase cassette) to obtain pCAMBIA2300-1. SEQ ID NO: 18:
GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO : 19：  GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO : 19:
ATCCAGATCTAGATCCGGTGCAGATTATTTG ATCCAGATCTAGATCCGGTGCAGATTATTTG
SEQ ID NO: 20禾 P SEQ ID NO: 21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 PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 20禾 P SEQ ID NO: 21各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环 (94°C 变性 30 s， 58 °C退火 30 s， 72 °C 延伸 30 s)， 72 °C 延伸 10 min。 通过 Sacl、 EcoRI 酶切后将所得 PCR 产物连接到 pCAMBIA2300-lCpromega T4 连接酶盒）获得 pCAMBIA2300-2。 SEQ ID NO: 20 and P SEQ ID NO: 21 Amplifies Tnos with pBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5 ><PS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ PrimeSTAR, 10 μΜ primer SEQ ID NO: 20 and P SEQ ID NO: 21 each 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min. The resulting PCR product was digested with Sacl, EcoRI and ligated into the pCAMBIA2300-l Cpromega T4 ligase cassette) to obtain pCAMBIA2300-2.
SEQ ID NO : 20： SEQ ID NO: 20:
AAGdCTCGAATTTCCCCGATCGTTCAAA  AAGdCTCGAATTTCCCCGATCGTTCAAA
SEQ ID NO : 21： SEQ ID NO: 21:
CAGAA rrCCCAGTGAATTCCCGATCTAGTA  CAGAA rrCCCAGTGAATTCCCGATCTAGTA
SEQ ID NO: 22和 SEQ ID NO: 23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 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM 的 dNTP， 1.0 μΐ 稀释 50 倍的 pCAMBIA2300 质粒， 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO :22禾 P SEQ ID NO :23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， 50°C退火 30 s， 72 °C 延伸 30 s；)， 72 °C 延伸 10 min。 通过 HindIII、 Pstl酶切后将所得 PCR产物连接 到 （连接方法同上） pCAMBIA2300-2获得 pCAMBIA2300-3 SEQ ID NO: 22 and SEQ ID NO: 23 amplify the Arabidopsis thaliana 35S promoter using the pCAMBIA2300 plasmid as a template. PrimeSTAR HS DNA polymerase using TaKaRa. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ diluted 50-fold pCAMBIA2300 plasmid, 1.0 μΐ PrimeSTAR, 10 μΜ primer SEQ ID NO: 22 and P SEQ ID NO: 23 each 2.0 11, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 min, 33 cycles (denaturation at 94 ° C for 30 s, annealing at 50 ° C for 30 s, Extend at 72 °C for 30 s;), extend at 72 °C for 10 min. The resulting PCR product was ligated by HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3
SEQ ID NO : 22： SEQ ID NO: 22:
ACTA^CHATGGTGGAGCACGACACTCT  ACTA^CHATGGTGGAGCACGACACTCT
SEQ ID NO : 23：  SEQ ID NO: 23:
TGA d AGAGATAGATTTGTAGAGAGAGAC  TGA d AGAGATAGATTTGTAGAGAGAGAC
SEQ ID NO: 24和 SEQ ID NO: 25扩增 GhHDbZIP-1 (模板是实施例 2所获得的 阳性 GhHDbZIP-1-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 GhHDbZIP-l-pGEM质粒， 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase、 10 μΜ的引物 SEQ ID NO :24禾 P SEQ ID NO: 25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， 58 °C退火 30 s， 72 °C 延伸 lmin30s), 72 °C 延伸 5 min。 通过 BamHI、 Sacl酶切后将所得 PCR产物连接 （连接方 法同上） 到 pCAMBIA2300-3， 获得植物表达载体 35S-GhHDbZIP-l-2300。 SEQ ID NO : 24： SEQ ID NO: 24 and SEQ ID NO: 25 Amplified GhHDbZIP-1 (template is the positive GhHDbZIP-1-pGEM plasmid obtained in Example 2), PfuUltra II Fusion HS DNA Polymerase using stratagene 50 μΐ PCR reaction system: 5 Μΐ lO PfuUltra II reaction Buffer, 0.5 μl 25 mM dNTP, 2.0 μΐ GhHDbZIP-l-pGEM plasmid, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase, 10 μΜ primer SEQ ID NO: 24 and P SEQ ID NO: 25 each 2.0 11, and 37.5 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 95 °C for 2 min, 35 cycles (denaturation at 95 °C for 25 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min 30 s), extension at 72 °C for 5 min. The resulting PCR product was ligated by BamHI and Sacl (ligation method) to pCAMBIA2300-3 to obtain a plant expression vector 35S-GhHDbZIP-l-2300. SEQ ID NO: 24:
GAGGA ΓίΤ TTGATGAATTTTGGGGGGTT  GAGGA ΓίΤ TTGATGAATTTTGGGGGGTT
SEQ ID NO : 25：  SEQ ID NO: 25:
AAGdCTCCATCAACTTTCGCATTGAAAG 实施例 4 35S-GhHDbZIP-l-2300表达载体转化农杆菌  AAGdCTCCATCAACTTTCGCATTGAAAG Example 4 35S-GhHDbZIP-l-2300 Expression Vector Transformation of Agrobacterium
农杆菌 LBA4404 (购自 Biovector Science Lab,Inc) 感受态细胞的制备： 提前 1-2天 将农杆菌 LBA4404在含 50 g/ml利福平和 50 g/ml链霉素的 LB固体培养基上划单斑接 种， 28°C培养 1至 2天。 挑取单菌落接种于 5 1^含50 4§/1^利福平和50 4§/1^链霉素的 LB液体培养基中， 28°C下摇动培养过夜 (约 12-16 h)至 OD6QQ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 h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 重悬浮菌体, 4°C下 4000 g离心 10 min， 收集沉淀； 用冰预冷的 10%甘油重复洗 3-4次； 加入适量冰浴预冷的 10%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。 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 in 5 1^ LB liquid medium containing 50 4 § /1^ rifampicin and 50 4 § /1^ streptomycin, and cultured overnight (about 12-16 h) at 28 °C. The OD 6QQ value was 0.4, and a seed bacterial liquid was formed. 5 ml of activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium of the same concentration of antibiotics, and cultured at 28 ° C for 2 to 2.5 h to OD 6 . . =0.8. The ice bath solution was shaken for 10 min every 3 min to allow the bacteria to enter the dormant state evenly. Centrifuge at 4000 g for 10 min at 4 ° C, discard the supernatant; add a certain amount of ice-cold 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 ° C, collect the precipitate; pre-cool with ice 10 Repeat 3-4 times of glycerol washing; resuspend the bacterial pellet by adding 10% glycerol pre-cooled in an appropriate amount of ice bath, dispense it at 40 μΐ/tube, and store at -70 °C. use.
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-GhHDbZIP-l-2300质粒， 混匀后冰浴约 10 min。 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 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， 28°C， 225 rpm培养 1 h。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 μ§/ιη1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 Transformation of Agrobacterium: The competent cells were thawed on ice, and 1 μΐ of the positive 35S-GhHDbZIP-l-2300 plasmid obtained in Example 3 was added to 40 μΐ of competent cells, and the mixture was mixed and ice bathed for about 10 min. 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. Using a 0.1 cm size electric shock cup, the MicroPuMUer (purchased from bio-rad) program is set to "Agr" and the shock is applied once. The electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added. Quickly and gently spread the cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h. 100-200 μL of bacterial solution is applied to the corresponding resistant screening medium plate (LB solid medium containing 50 μ § /ιη1 rifampicin, 50 μ § /ιη1 streptomycin, 50 μ § /ιη1 Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Obtaining transgenic Arabidopsis thaliana using Agrobacterium-mediated transformation
待转化植株培养： 拟南芥种子 （哥伦比亚型， 来自美国俄亥俄州立大学的拟南芥生 物资源中心）播种在泥炭土中， 经 4°C低温处理 3天后， 置于 23 °C、 16h光照 /8h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， 16h光照 /8h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 l_10 cm高 （剪去第一个花序后 4_8 d) 时准备浸染。  Plants to be transformed: Arabidopsis seeds (Colombian type, Arabidopsis Bioresource Center, Ohio State University, USA) were sown in peat soil, treated at 4 ° C for 3 days, placed at 23 ° C, 16 h light / Germinated in a dark incubator at 8h. After 7-10 days, transplanted into a plastic pot with a diameter of 7.5 cm containing peat soil and vermiculite (3:1 by volume), 6 plants per pot, placed at 23 °C, 16h light/8h dark culture Growing in the box. 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. Every 3-4 weeks (or longer) as needed. In order to obtain more flower buds on each plant, when the first inflorescence of most plants is formed, the first inflorescence is cut off, and the apical dominance is removed, prompting the simultaneous emergence of multiple secondary inflorescences. Prepare for dip when most of the inflorescences are about l_10 cm high (4_8 d after the first inflorescence is cut).
农杆菌的培养： 取出实施例 4中保种的农杆菌阳性转化克隆的菌液活化后， 挑取农 杆菌单菌落接种到 10 mL无菌 LB液体培养基中 （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 r/ min振摇过夜培养。 再将所得到的菌液按 1%— 2%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 OD6QQ=1.8， 然后在 4 °C下 3000 r/min离心 15 min, 弃去上清液后用浸 染培养基 （该浸染培养基含有 5.0%的蔗糖和 0.05% (500 μΙΤί) 的 Silwet L-77) 重新悬 浮农杆菌， 悬浮至 OD6QQ约 0.80。 Culture of Agrobacterium: After removing the bacterial solution of the Agrobacterium-positive transformed 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 r/min 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% to 2%, and shaken at a constant temperature of 28 ° C to achieve the concentration of Agrobacterium to OD 6QQ = 1.8, and then at 4 Centrifuge at 3000 r/min for 15 min at °C, discard the supernatant and resuspend the Agrobacterium with the dipping medium (5.0% sucrose and 0.05% (500 μΙΤί) of Silwet L-77). Up to 0.80 to OD 6QQ .
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 h去掉覆盖。 正常培养植株， 植株 进一步生长 3— 5周， 直至角果变褐变干。 收获种子， 并将种子用离心管在 4 °C下干燥贮 存。 Inflorescence dip dyeing: The above Agrobacterium-containing dyeing medium was added to a large-mouth container, and 200-300 mL of the Agrobacterium-containing dyeing medium was added to each container having a diameter of 9 cm for dip dyeing. Invert the plant to make the ground tissue Immerse 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 plastic wrap to moisturize, then placed in low light or dark places overnight, taking care to prevent direct sunlight from the plants. The cover was removed about 12-24 hours after the treatment. 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.
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 1 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: 16： 和 SEQ ID NO: 17扩增 GhHDbZIP-1 ： ( 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: 16和 SEQ ID NO: 17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 30 s, 54°C退火 30 s， 72 °C 延伸 2 min)， 72 °C 延伸 10 min)， 将 PCR鉴定为阳性的植株 进行编号 （T1D1-T1D16), 并保存。 实施例 6 过表达 GhHDbZIP-1的转基因拟南芥 T1代植株的耐旱模拟实验及功能鉴定 灭过菌的蛭石用 1/2MS培养基浸透。 T1D1-T1D6及对照拟南芥种子分别播种在蛭石 上，每盆播种 10颗种子， 25°C、 10小时光培养 /14小时暗培养循环，每 7天浇一次 1/2MS, 培养 20天之后， 每盆保留大小较一致的 4棵苗， 用于干旱实验。 转基因拟南芥、 对照拟 南芥干旱 14天 (不浇水)， 25°C、 10小时光培养 /14小时暗培养循环。 T1代转基因植株（T0 代转基因植株的种子长成的植株） 的抗旱性鉴定表明， 对照植株都萎蔫严重， 而 T1D1、 T1D2、 T1D3、 T1D4、 T1D5、 T1D6 六个株系共 24 (每株系各 4棵） 拟南芥中 21能够 存活并继续生长显现出明显的耐旱性 （参见图 3a和 3b， 以 T1D2、 T1D6为例， T1D1、 T1D3、 T1D4、 T1D5的结果与 T1D2、 T1D6 类似， 在此未示出）。 实施例 7 干旱胁迫后 ABA变化的测定 Screening of transgenic seeds: Prepare an aqueous solution containing 1/4 MS of large elements, add 0.8% agar powder, heat in a microwave oven until the agar is completely dissolved, and cool to about 50 °C, add the required amount of card with a final concentration of 50 mg 1 Natamycin, shake well, pour 25 mL into each dish, and set the table to cool and solidify before seeding. Pour the weighed seeds on a piece of plain copy paper, tap the copy paper with your fingers, evenly sow the seeds on the agar gel, cover the Petri dish, and cool the solution at 4 °C for 72 h, then move to Germination was carried out in an incubator at 23 °C, 16h light/8h dark, and seed germination and seedling growth were regularly counted, and the resistant seedlings were transplanted into the nutrient soil in time. After transplanting, the soil moisture is added to the water in time. The nutrient solution is properly watered during the growth period. Take 0.1 g of Arabidopsis leaves grown for 20 days, extract DNA, and amplify GhHDbZIP-1 with SEQ ID NO: 16: and SEQ ID NO: 17 (50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ DNA, 1.0 μΐ Ex Taq, 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: 94 °C predenaturation 5 Min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min), numbering plants identified as positive by PCR (T1D1-T1D16) And save. Example 6 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis thaliana T1 plants overexpressing GhHDbZIP-1. The sterilized vermiculite was soaked in 1/2 MS medium. T1D1-T1D6 and control Arabidopsis seeds were planted on vermiculite, 10 seeds per pot, 25 ° C, 10 hours light culture / 14 hours dark culture cycle, 1/2 MS every 7 days, after 20 days of culture 4 seedlings of uniform size were kept in each pot for drought experiments. Transgenic Arabidopsis thaliana, control Arabidopsis thaliana drought for 14 days (no watering), 25 ° C, 10 hours light culture / 14 hours dark culture cycle. The drought resistance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that the control plants were wilting, while T1D1, T1D2, T1D3, T1D4, T1D5, T1D6 had 24 strains (each line). In each of the four Arabidopsis thaliana, 21 survived and continued to grow to show significant drought tolerance (see Figures 3a and 3b. Taking T1D2 and T1D6 as examples, the results of T1D1, T1D3, T1D4, and T1D5 are similar to those of T1D2 and T1D6. Not shown here). Example 7 Determination of ABA change after drought stress
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（T1D1、 T1D2、 T1D3、 T1D4、 T1D5、 T1D6)及对照植株 （CK1、 CK2) 叶片各 0.2 g左右，用中国农业大学作物化控研究中心制备的试剂盒测定 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 GhHDbZIP-1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 GhHDbZIP蛋白表达 ABA is recognized as a plant hormone associated with stress, which can act as a signaling molecule to regulate multiple adverse temptations. The expression of the derivative gene, thereby improving the plant's resistance to stress. We took about 0.2 g of transgenic plants (T1D1, T1D2, T1D3, T1D4, T1D5, T1D6) and control plants (CK1, CK2) under drought stress for 10 days and normal growth conditions, and used the Crop Control Research Center of China Agricultural University. The prepared kit measures the ABA content (see Figure 4). The results showed that the ABA content of transgenic plants was higher than that of the control (CK1, CK2) under drought treatment and control conditions, which indicated that GhHDbZIP-1 gene could positively regulate the endogenous ABA content of plants. Example 8 Validation of GhHDbZIP protein expression at the transcriptional level
分别取对照拟南芥植株、 耐旱转基因拟南芥 T1 代植株 （分别属于 T1D1、 T1D2、 T1D3、 T1D4、 T1D5、 T1D6六个株系）、 和不耐旱转基因拟南芥 Tl代植株的干旱 10天 的叶片各 0.05 g， 用植物 RNA提取试剂盒 （invitrogen) 提取的总 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 μβ总 RNA作为模板，反转录引物 SEQ ID NO: 7)。通过 SEQ ID NO: 16和 SEQ ID NO: 17扩增 GhHDbZIP, 检测 HDbZIP蛋白相对表达情况。 Control Arabidopsis thaliana plants, drought-tolerant transgenic Arabidopsis thaliana T1 plants (six T1D1, T1D2, T1D3, T1D4, T1D5, T1D6, respectively), and drought-tolerant transgenic Arabidopsis thaliana T1 plants Total RNA extracted by a plant RNA extraction kit (invitrogen) at 0.05 g each for 10 days. The absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations. Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (2 μ β total RNA as a template, reverse transcription primer SEQ ID NO: 7). The relative expression of HDbZIP protein was detected by amplifying GhHDbZIP by SEQ ID NO: 16 and SEQ ID NO: 17.
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和 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 min， 29个循环 （ 94°C 变性 30 s， 54 °C 退火 30 s， 72 °C 延伸 2min) ， 72 °C 延伸 10 min。  PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template. 50 μl ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μl, and 30 μΐ Double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 29 cycles (denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
产物电泳结果如图 5所示： M为 DNA Ladder Marker ( DL5000, TakaRa) ， 1-5 为耐旱转基因拟南芥 Tl代植株（依次为： T1D1、 T1D2、 T1D3、 T1D4、 T1D5 ) ， 6-8 为非转基因拟南芥对照， 9-1 1为不耐旱转基因拟南芥 T1代植株。 图中所示 PCR产物 电泳条带大小与 GhHDbZIP-1的大小一致 （约 2.4 Kbp ) 。 结果表明， 对照拟南芥没 有 GhHDbZIP-1转录， 耐旱转基因拟南芥 T1代植株中 GhHDbZIP-1的转录较强，不耐 旱转基因拟南芥 T1代植株没有转录或转录很弱。  The electrophoresis results of the product are shown in Figure 5: M is the DNA Ladder Marker (DL5000, TakaRa), and 1-5 is the drought-tolerant transgenic Arabidopsis T1 plant (in order: T1D1, T1D2, T1D3, T1D4, T1D5), 6- 8 is a non-transgenic Arabidopsis control, and 9-1 1 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant. The size of the electrophoresis band of the PCR product shown in the figure is consistent with the size of GhHDbZIP-1 (about 2.4 Kbp). The results showed that there was no GhHDbZIP-1 transcription in Arabidopsis thaliana, and the transcription of GhHDbZIP-1 in the drought-tolerant transgenic Arabidopsis thaliana T1 plants was stronger, and the drought-tolerant transgenic Arabidopsis thaliana T1 plants were not transcriptionally or transcriptionally weak.

Claims

权 利 要 求 书 Claim
1. 棉花的一个同源异型-亮氨酸拉链蛋白的编码基因， 其序列为 SEQ ID NO: 2。A gene encoding a homeobox-leucine zipper protein of cotton having the sequence SEQ ID NO: 2.
2. 一种重组表达载体，其含有权利要求 1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。 2. A recombinant expression vector comprising the gene of claim 1 and the nucleotide sequence of the gene operably linked to an expression control sequence of the expression vector.
3. 权利要求 2所述的载体， 其为附图 2所示的 35S-GhHDbZIP-l-2300载体。 3. The vector of claim 2 which is the 35S-GhHDbZIP-1-23 carrier shown in Figure 2.
4. 一种重组细胞，其含有权利要求 1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。 A recombinant cell comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3; preferably, the recombinant cell is a recombinant Agrobacterium cell.
5. 一种改善植物耐旱性的方法， 包括： 将权利要求 1所述的基因或者权利要求 2 或 3所述的重组表达载体导入植物或植物组织并使所述基因表达； 优选地， 所述植物 是拟南芥。  A method for improving drought tolerance of a plant, comprising: introducing the gene of claim 1 or the recombinant expression vector of claim 2 or 3 into a plant or plant tissue and expressing the gene; preferably, The plant is Arabidopsis.
6. 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 1所述的基因或者权利要求 2或 3所述的重组表达载体的植物或植物组织。  A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3 under conditions effective to produce a plant.
7. 权利要求 6所述的方法， 其中所述植物是拟南芥。  7. The method of claim 6, wherein the plant is Arabidopsis thaliana.
8. 权利要求 1所述的基因、权利要求 2或 3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。  The gene of claim 1, the recombinant expression vector of claim 2 or 3, or the recombinant cell of claim 4 for use in improving drought tolerance of a plant and for use in plant breeding.
9. 权利要求 8所述的用途， 其中所述植物是拟南芥。  9. The use of claim 8, wherein the plant is Arabidopsis thaliana.
10. 权利要求 1所述的基因编码的蛋白， 其氨基酸序列如 SEQ ID NO: 1所示。  The gene-encoded protein according to claim 1, which has an amino acid sequence as shown in SEQ ID NO: 1.
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