WO2014169472A1 - Cotton leucine zipper protein bzip-3, coding genes of same, and application thereof - Google Patents

Cotton leucine zipper protein bzip-3, coding genes of same, and application thereof Download PDF

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WO2014169472A1
WO2014169472A1 PCT/CN2013/074402 CN2013074402W WO2014169472A1 WO 2014169472 A1 WO2014169472 A1 WO 2014169472A1 CN 2013074402 W CN2013074402 W CN 2013074402W WO 2014169472 A1 WO2014169472 A1 WO 2014169472A1
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plant
seq
gene
expression vector
cotton
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PCT/CN2013/074402
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French (fr)
Chinese (zh)
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陈文华
孙超
崔洪志
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创世纪转基因技术有限公司
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Priority to CN201380074537.6A priority Critical patent/CN105189537A/en
Priority to PCT/CN2013/074402 priority patent/WO2014169472A1/en
Publication of WO2014169472A1 publication Critical patent/WO2014169472A1/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 their coding genes and applications, and more particularly to a cotton-derived leucine zipper protein bZIP-3 and a gene encoding the same, and its use in breeding transgenic plants with improved drought tolerance.
  • 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.
  • 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 cloned a coding gene for a leucine zipper protein (designated herein as bZIP-3) of cotton using SSH (suppression subtractive hybridization) in combination with RACE (rapid amplification of cDNA ends) and determined Its DNA sequence. Moreover, it was found that when introduced into the recipient plant and overexpressed, the drought tolerance of the transgenic plants was significantly improved, and And these traits can be stably inherited.
  • a first aspect of the invention provides a coding gene for a leucine zipper egg bZIP-3 of cotton (designated herein as GhbZIP-3) having the sequence SEQ ID NO: 2.
  • a second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, preferably, the expression vector is PCAMBIA2300 And the nucleotide sequence of the gene is operably linked to the expression control sequence of the recombinant expression vector; preferably, the recombinant expression vector is 35S-G)Z/P-3- as shown in FIG. 2300 carrier.
  • 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 plant early tolerance and for plant breeding Use;
  • the plant is Arabidopsis thaliana.
  • the seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, the amino acid sequence of which is shown in SEQ ID NO: 1.
  • Figure 1 is a construction flow of the plant expression vector C35S-G)Z/P-3-2300; of the G)Z/P-3 gene (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector (35S-GMZ/P-3-2300) of the GhbZIP-3 gene.
  • FIG. 3 is a drought tolerance simulation experiment of T2 transgenic Arabidopsis plants (Fig., T2F1; T2F5) of G)Z/P-3 gene and non-transgenic Arabidopsis plants (Fig., CK1, CK2) as control result.
  • Fig. 3a is an Arabidopsis plant that grows normally for 20 days
  • Fig. 3b shows an Arabidopsis plant that has been treated for 14 days after normal growth for 14 days).
  • Figure 4 is a molecular detection verification result of transcriptional level of T2 transgenic Arabidopsis plants and non-transgenic control plants GhbZIP-3 gene by reverse transcription PCR.
  • M is DNA Ladder Marker (DL2000, TakaRa)
  • 1-6 is a drought-tolerant transgenic Arabidopsis thaliana T2 plant (in order: T2FK T2F2, T2F3, T2F4, T2F5, T2F6)
  • 7-10 is a transgenic Arabidopsis thaliana T2 plant with insignificant drought tolerance
  • 11-14 is a non-transgenic control Arabidopsis plant.
  • BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples. .
  • the SSH library (subtractive library) was constructed by inhibition subtractive hybridization according to the method described in Clontech's PCR-selectTM cDNA Subtraction Kit kit.
  • the mRNA of the leaves of the drought-treated cotton seedlings was used as a sample (Tester mRNA) during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a control (Driver mRNA).
  • the specific steps are as follows:
  • the above 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 16 h light / 8 h dark, and was usually 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 two of the seedlings were cut in time, and the liquid nitrogen was quickly used. After freezing, store in a -70 ° C refrigerator.
  • the cotton leaves of the control and drought-treated groups were each 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 was measured by HITACHI's UV spectrophotometer U-2001.
  • the ratio of OD 260 / OD 280 was 1.8-2.0, indicating that the total RNA purity was higher; 1.0% agarose gel was used.
  • Gel electrophoresis detects total RNA integrity, bright 28S bands The degree is about 2 times that of the 18 S band, indicating that the integrity of the RN A is good.
  • mRNA was isolated using Qiagen's purification of poly A+ RNA from total RNA.
  • Suppression subtractive hybridization was performed as indicated by Clontech's PCR-selectTM cDN A Subtraction Kit kit instructions.
  • the Driver mRNA and Tester mRNA were reverse transcribed to obtain a double-stranded cDNA, and then subtracted hybridization using 2 Tester cDNA and 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
  • this experiment uses the endonuclease Haelll
  • the above steps were carried out by digestion of the Tester cDNA and the Driver cDNA, followed by two positive subtractive hybridizations and two inhibitory PCR amplifications, and finally the second inhibitory PCR products of the two sets of forward subtractive hybridization cDNA fragments were combined.
  • the second PCR product of the above combined positive subtractive hybridization cDNA fragment purified using QIAquick PCR Purification Kit, purchased from Qiagen
  • pGEM-T Easy kit purchased from Promega
  • the specific steps are as follows: The following components are sequentially added to a 200 ⁇ PCR tube: the second inhibitory PCR product of the purified combined positive subtractive hybridization cDNA fragment 3 ⁇ 1, 2 ⁇ 4 DNA ligase buffer 5 ⁇ l of liquid, pGEM-T Easy vector 1 ⁇ l, T4 DNA ligase 1 ⁇ , and ligated overnight at 4 °C.
  • sequence was SEQ ID No: 3.
  • sequence analysis indicated that the protein encoded by the sequence belonged to the leucine zipper protein, and the SEQ ID No: 3 corresponding to the SEQ ID No: 3
  • the full-length coding gene was named GhbZIP-3, and its corresponding protein was named bZIP-3.
  • SEQ ID No: 3 is the coding gene
  • GhB98 GSP1 SEQ ID NO: 4:
  • GhB98 GSP2 SEQ ID NO: 5:
  • GhB98 GSP3 SEQ ID NO:6:
  • the kit comes with universal primers:
  • AAP SEQ ID NO: 7:
  • the GGCCACGCGTCGACTAGTAC experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • GhB98 GSP1 (SEQ ID NO: 4) was used as a reverse transcription primer, and reverse transcription was performed using cotton mRNA as a template to obtain a cDNA template. Then, according to the procedure in the above 5' RACE kit instructions, Poly C tail was added to end the tail. The latter product was subjected to the first round of PCR amplification using the primers of SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (the kit is self-contained, and I is hypoxanthine modified a, c, g or t ), Specific steps are as follows:
  • PCR reaction system 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcription and Poly C tail cDNA, 1.0 ⁇ Ex Taq (purchased from TAKARA), 10 ⁇ primer SEQ ID NO: 4 and SEQ ID NO: 7 each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (94 denaturation for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50-fold with double distilled water, and 2.0 ⁇ L was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 5 and the general primer SEQ ID NO: 8, and the specific steps were as follows:
  • PCR product 1.0 l Ex Taq, 10 ⁇ primers SEQ ID NO: 5 and SEQ ID NO: 8 each 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (94 denaturation for 45 s, annealing at 56 °C for 45 s, 72 V for 1 min), extension at 72 °C for 10 min.
  • a band of about 750 bp in size from the second round of PCR product (GEL extraction kit from OMEGA) was recovered and ligated into pGEM-T Easy vector, which was then transformed into E.
  • a pair of primers were designed according to the sequence of SEQ ID NO: 10 as follows:
  • GhbZIP-3F SEQ ID NO: 11:
  • GhbZIP-3R SEQ ID NO: 12:
  • the GhbZIP-3 full-length coding sequence was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
  • Stratagene's PfuUltra II Fusion HS DNA Polymerase was used for PCR reaction using cotton cDNA as a template.
  • PCR reaction conditions pre-denaturation at 95 °C for 2 min, 35 cycles (denaturation at 95 °C for 25 s, annealing at 53 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 5 min.
  • PCR amplification product plus A tail PCR product plus 2.5 volumes of absolute ethanol, placed at -20 ° C for 10 min, centrifuged, supernatant, dried, dissolved in 21 ⁇ l double distilled water. Add 2.5 ⁇ ⁇ Buffer, 0.5 ⁇ 5 mM dATP, 1.0 l Ex Taq. Reaction conditions: 70 ° C reaction for 30 min. The obtained 1200 bp DNA fragment was recovered (Omega Recovery Kit) and ligated to the pGEM T-easy vector (obtained)
  • GhbZIP-3 -pGEM plasmid was then transformed into E. coli JM109 competent cells and screened on LB solid medium containing 50 g/mL ampicillin (method supra). Eight white colonies were randomly picked and inoculated 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.
  • the primers SEQ ID NO: 1 1 and SEQ ID NO: 12 were used for PCR amplification (the reaction system and the reaction conditions are the same as above), and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • the sequence is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is SEQ ID NO: 1.
  • OiOiOOiDDV ⁇ DO ⁇ iDDiO i ⁇ ii ⁇ DD ⁇ D 0 ⁇ DO ⁇ DiiDO ⁇ DiDWiiO ⁇ DD ⁇ DO ⁇ Di ⁇ T99
  • OdOSi SOVO ( ⁇ ⁇ a ⁇ OSdOl ⁇ 3 ⁇ 0 ⁇ ⁇ 33 ⁇ 13 1 ⁇ OOHOdW aiV IZl aiOIS ⁇ a SI aHMAaaAi ⁇ H Sll ⁇ dl ⁇ 1S£) ⁇ OAOdSVWiOi 33 ⁇ 3 ⁇ 3 ⁇ 4 ⁇ 19 Oooaoosdd dNS Jsrai flat ⁇
  • the plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ⁇ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. .
  • the 35S promoter containing the double enhancer and the Tnos terminator were selected as promoters and terminators of the G)Z/P-3 gene, respectively.
  • the construction process is shown in Figure 1.
  • Primer SEQ ID NO: M SEQ ID NO: 15 was used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ PCR reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ pBI121 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 14 and SEQ ID NO: 15 each 2.0 ⁇ l, and 31 ⁇ double Steamed water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was digested with EcoRI and Bglll and ligated into lj pCAMBIA2300 (Promega, T4 ligase cassette) to obtain pCAMBIA2300-1.
  • Primers SEQ ID NO: 16 and SEQ ID NO: 17 were used to amplify Tnos using pBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ PCR reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ pBI121, 1.0 ⁇ Prime STAR, 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 ⁇ 1, and 31 ⁇ Double steamed water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was digested with Sall, EcoRI and ligated into pCAMBIA2300-1 (Promega T4 ligase cassette) to obtain pCAMBIA2300-2.
  • the 35S promoter was amplified using the primers SEQ ID NO: 18 and SEQ ID NO: 19 using the pCAMBIA2300 plasmid as a template.
  • PrimeSTAR HS DNA polymerase using TaKaRa 50 ⁇ PCR reaction system: 10 l 5 > ⁇ PS Buffer 3 ⁇ 2.5 mM dNTPs, 1.0 ⁇ diluted 50-fold pCAMBIA2300 plasmid, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 18 and P SEQ ID NO: 19 each 2.0 ⁇ l, and 31 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (denaturation at 94 ° C for 30 s, annealing at 50 ° C for 30 s, extension at 72 ° C for 30 s), extension at 72 ° C for 10 min.
  • the resulting PCR product was ligated by HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3
  • the GMZH ⁇ -pGEM plasmid containing the full-length GhbZIP-3 gene in Example 2 was used as a template to amplify GMZH ⁇ with restriction sites at both ends. gene. Stratagene's PfuUltra II Fusion HS DNA Polymerase was used.
  • PCR reaction system 5 ⁇ lO PfuUltra II reaction Buffer 0.5 ⁇ 125 mM dNTP, 2.0 ⁇ GhbZIP-3-pGEM plasmid, 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase 10 ⁇ primers SEQ ID NO: 20 and SEQ ID NO: 21 each of 2.0 ⁇ l, and 37.5 ⁇ of double distilled water.
  • PCR reaction conditions Pre-denaturation at 95 °C for 2 min, 35 cycles (denaturation at 95 °C for 25 s, annealing at 58 °C for 30 s, extension at 72 °C for 45 s), extension at 72 °C for 5 min.
  • the obtained PCR product was ligated (the ligation method was the same as above) to pCAMBIA2300-3, and the plant expression vector 35S-GhbZIP-3-2300 was obtained (Fig. 2).
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was spotted on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, 28 V is incubated for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin and 50 ⁇ ⁇ / ⁇ 1 streptomycin, and cultured overnight (about 12-16 h) to OD 6 at 28 °C. The K) value is 0.4, and a seed bacterial liquid is formed.
  • Transformation of Agrobacterium Melt LBA4404 competent cells on ice, add 1 ⁇ of the positive 35S-G)Zff-3-2300 plasmid obtained in Example 3 to 40 ⁇ of the competent cells, mix and mix, then ice bath 10 min.
  • the mixture of the competent cells after the ice bath and the positive 35S-G)Z/P-3-2300 plasmid was transferred to an ice-cold electric shock cup with a micropipette, and tapped to bring the suspension to the bottom. , be careful not to have 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 program of MicroPuMUer purchased from Bio-Rad
  • the program of MicroPuMUer was set to "Agr" and the shock was applied once.
  • the suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h.
  • 100-200 ⁇ L of bacterial solution is applied to the corresponding resistant screening medium plate (LB solid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin, 50 ⁇ ⁇ / ⁇ 1 streptomycin, 50 ⁇ ⁇ / ⁇ 1 Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use.
  • LB solid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin, 50 ⁇ ⁇ / ⁇ 1 streptomycin, 50 ⁇ ⁇ / ⁇ 1 Kanamycin
  • Example 5 Agrobacterium-mediated transformation was used to obtain transgenic Arabidopsis thaliana
  • Plant to be transformed Arabidopsis seeds (Colombian type, Arabidopsis Bioresource Center from Ohio State University) 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. Before transplanting, 40 ml of 1/2 MS liquid medium was poured per pot. After transplanting, the soil moisture was added to the soil in time. Properly water 1/2 MS liquid medium during growth. Every 3-4 weeks (or longer) as needed.
  • the first inflorescence of most plants is formed, the first inflorescence is cut to relieve the apical dominance and promote the simultaneous emergence of multiple secondary inflorescences.
  • most of the inflorescences are about 1 to 10 cm high (4 to 8 days after the first inflorescence is cut), they are ready to be infiltrated.
  • Infiltration of inflorescences Adding the above-mentioned Agrobacterium-containing dyeing medium to a large-mouth container, each container having a diameter of 9 cm 200-300 mL of the Agrobacterium-containing dyeing medium was added for dip dyeing. Invert the plants so that the aboveground tissues are completely immersed in the Agrobacterium suspension for 3-5 s and gently agitated. 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. T1 generation transgenic seeds were harvested and stored in a centrifuge tube at 4 °C.
  • Transgenic seed screening Prepare 1/2MS liquid medium, add 0.8% (w/v) agar powder, heat in a microwave oven until the agar is completely dissolved, cool to about 50 °C, add kanamycin to a final concentration of 50 mg -L" 1 , pour 25 mL into each dish after shaking, set the solid medium obtained after cooling and solidification on the test bench for seeding. Pour the weighed seeds on a piece of plain copy paper, lightly with your fingers Knock on the copy paper, soak the seeds evenly on the agar gel, cover the Petri dish cover, cool it in a refrigerator at 4 °C for 72 h, then transfer to a 23 V, 16 h light/8 h dark incubator to germination, and periodically count the seed germination.
  • 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 1 min), 72 °C extension for 7 min), the plants identified as positive by PCR were numbered, and the plants were cultured normally, and the plants were further grown for 3-5 weeks until the pods became brown and dried.
  • Harvested T2 transgenic seeds No.: T2F1-T2F16
  • the seeds were dried and stored in a centrifuge tube at 4 ° C.
  • Example 6 Drought-tolerant simulation experiment and functional identification of transgenic Arabidopsis thaliana T2 plants overexpressing GhbZIP-3 1/2MS of sterilized vermiculite The liquid medium was soaked. The transgenic seeds numbered T2F1-T2F8 obtained in Example 5 and the control Arabidopsis thaliana (non-transgenic) seeds were separately sown on the vermiculite, and 10 seeds were seeded per pot, 25 ° C, 10 h. Light culture/14 h dark culture cycle, 1/2 MS liquid medium was poured every 7 days, after 20 days of culture, positive plants were screened with 2000 PPM kanamycin coated leaves, and 4 Canames with uniform size were retained per pot. Tyrosine resistant seedlings, used in drought experiments.
  • the drought resistance of T2 transgenic plants showed that the control plants were wilting, while T2F1, T2F2 T2F3 T2F4, T2F5, Twenty-two transgenic lines of T2F6, T2F7 and T2F8 (4 in each line) 30 of the Arabidopsis thaliana survived and continued to grow with significant drought tolerance (see Figures 3a and 3b, with T2F1).
  • T2F5 is taken as an example.
  • T2F2 T2F3 T2C 4 T2F6 and T2F7 T2F8 are similar to those of T2F1 and T2F5, which are not shown here).
  • Example 7 Validation of GhbZIP-3 Gene Expression at Transcriptional Level
  • RNA extraction kit Invitrogen
  • the absorbance values of the total RNA obtained at 260 nm and 280 nm were measured by HITACHI's ultraviolet spectrophotometer U-2001, and the respective RNA concentrations were calculated.
  • Reverse transcription was carried out according to the method shown by Invitrogen Reverse Transcription Kit Superscript III Reverse Transcriptase (2 ⁇ g of total RNA as a template, reverse transcription of SEQ ID NO: 13).
  • the relative expression of bZIP-3 protein was examined by amplifying GhbZIP-3 by SEQ ID NO: 11 and SEQ ID NO: 12.
  • PCR was carried out using TaKaRa's PrimeSTAR HS DNA polymerase using the above reverse transcribed cDNA as a template.
  • 50 ⁇ l ⁇ Reaction system 10 ⁇ 5 > ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 l PrimeSTAR, 10 ⁇ primer SEQ ID NO: 1 1 and P SEQ ID NO: 12 each 2.0 ⁇ l, And 30 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 29 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 V for 45 s), extension at 72 °C for 10 min.
  • M is DNA Ladder Marker (DL2000, TakaRa), and 1-6 is a significantly drought-tolerant transgenic Arabidopsis thaliana T2 plant (in order: T2F1, T2F2 T2F3 T2F4, T2F5, T2F6); -10 is a T2 generation of transgenic Arabidopsis thaliana plants that are not significantly drought tolerant; 11-14 is a non-transgenic Arabidopsis control.
  • the size of the electrophoresis band of the PCR product shown in the figure is the same as the size (about 1200 bp).

Abstract

The present invention relates to a plant protein, the coding genes of same, and an application thereof, and specifically relates to a leucine zipper protein sourced from cotton, bZIP-3, the coding genes of same, and an application thereof in cultivating a transgenic plant of increased drought tolerance.

Description

一种棉花亮氨酸拉链蛋白 bZIP-3及其编码基因与应用  Cotton leucine zipper protein bZIP-3 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亮氨 酸拉链蛋白 bZIP-3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。 TECHNICAL FIELD The present invention relates to plant proteins and their coding genes and applications, and more particularly to a cotton-derived leucine zipper protein bZIP-3 and a gene encoding the same, and its use in breeding transgenic plants with improved drought tolerance. 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、 优质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 high quality 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 ^言， 由于其机制十分复杂，许多植物对逆境的生物化学和生理学响 应机制仍有待深入研究。 在抗逆应答基因的功能及表达调控方面的研究将为植物抗逆相 关的信号传递途径之间的联系以及整个信号传递网络系统的研究提供重要的基础。 发明内容 本发明人利用 SSH (抑制差减杂交） 与 RACE (cDNA末端快速扩增） 相结合的方 法克隆了棉花的一个亮氨酸拉链蛋白（本文命名为 bZIP-3 )的编码基因，并测定了其 DNA 序列。 并且发现将其导入受体植物并超量表达后， 可显著改善转基因植株的耐旱性， 而 且这些性状可稳定遗传。 When plants are stressed by stress, they will respond accordingly to reduce or eliminate the damage caused by stress. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress remain to be further studied. Studies on the function and expression regulation of stress-responsive genes will provide an important basis for the link between plant stress-resistance-related signaling pathways and the study of the entire signaling network system. SUMMARY OF THE INVENTION The present inventors cloned a coding gene for a leucine zipper protein (designated herein as bZIP-3) of cotton using SSH (suppression subtractive hybridization) in combination with RACE (rapid amplification of cDNA ends) and determined Its DNA sequence. Moreover, it was found that when introduced into the recipient plant and overexpressed, the drought tolerance of the transgenic plants was significantly improved, and And these traits can be stably inherited.
本发明第一方面提供棉花的一个亮氨酸拉链蛋 bZIP-3 的编码基因 （本文命名为 GhbZIP-3) ， 其序列为 SEQ ID NO: 2。  A first aspect of the invention provides a coding gene for a leucine zipper egg bZIP-3 of cotton (designated herein as GhbZIP-3) having the sequence SEQ ID NO: 2.
本发明第二方面提供一种重组表达载体， 其含有本发明第一方面所述的基因， 其是 通过将所述基因插入到一种表达载体而获得的， 优选地， 所述表达载体是 PCAMBIA2300;并且所述基因的核苷酸序列与所述重组表达载体的表达控制序列可操作 地连接； 优选地， 所述重组表达载体为附图 2所示的 35S-G )Z/P-3-2300载体。  A second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, preferably, the expression vector is PCAMBIA2300 And the nucleotide sequence of the gene is operably linked to the expression control sequence of the recombinant expression vector; preferably, the recombinant expression vector is 35S-G)Z/P-3- as shown in FIG. 2300 carrier.
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早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 plant early tolerance and for plant breeding Use; Preferably, the plant is Arabidopsis thaliana.
本发明第七方面提供由本发明第一方面所述的基因编码的蛋白质， 其氨基酸序列如 SEQ ID NO: 1所示。 附图说明 图 1是 G )Z/P-3基因的植物表达载体 C35S-G )Z/P-3-2300;>的构建流程 （图 la-lb )。 图 2是 GhbZIP-3基因的植物表达载体 (35S-GMZ/P-3-2300)的质粒图。  The seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, the amino acid sequence of which is shown in SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a construction flow of the plant expression vector C35S-G)Z/P-3-2300; of the G)Z/P-3 gene (Fig. la-lb). Figure 2 is a plasmid map of the plant expression vector (35S-GMZ/P-3-2300) of the GhbZIP-3 gene.
图 3是 G )Z/P-3基因的 T2代转基因拟南芥植株（图中， T2F1 ; T2F5 )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 is a drought tolerance simulation experiment of T2 transgenic Arabidopsis plants (Fig., T2F1; T2F5) of G)Z/P-3 gene and non-transgenic Arabidopsis plants (Fig., CK1, CK2) as control result. (Fig. 3a is an Arabidopsis plant that grows normally for 20 days; Fig. 3b shows an Arabidopsis plant that has been treated for 14 days after normal growth for 14 days).
图 4是利用反转录 PCR对 T2代转基因拟南芥植株和非转基因对照植株种 GhbZIP-3 基因在转录水平上的分子检测验证结果。 M为 DNA Ladder Marker (DL2000,TakaRa), 1-6为耐旱效果显著转基因拟南芥 T2代植株（依次为： T2FK T2F2、 T2F3、 T2F4、 T2F5、 T2F6) ； 7-10为耐旱效果不显著的转基因拟南芥 T2代植株 ; 11-14为为非转基因的对照拟 南芥植株. 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。 Figure 4 is a molecular detection verification result of transcriptional level of T2 transgenic Arabidopsis plants and non-transgenic control plants GhbZIP-3 gene by reverse transcription PCR. M is DNA Ladder Marker (DL2000, TakaRa), 1-6 is a drought-tolerant transgenic Arabidopsis thaliana T2 plant (in order: T2FK T2F2, T2F3, T2F4, T2F5, T2F6); 7-10 is a transgenic Arabidopsis thaliana T2 plant with insignificant drought tolerance; 11-14 is a non-transgenic control Arabidopsis plant. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples. .
下面实施例中提到的未注明来源的限制性内切酶均购自 New England Biolabs公司。 实施例 1、 干旱胁迫下棉花 SSH文库构建：  The unrecognized restriction enzymes mentioned in the examples below were purchased from New England Biolabs. Example 1. Cotton SSH library construction under drought stress:
具体方法为：  The specific method is:
按照 Clontech公司的 PCR-select™ cDNA Subtraction Kit试剂盒说明书所示的方法通 过抑制差减杂交方法构建 SSH文库 （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 mRNA), 以未处理的棉花幼苗的叶片的 mRNA作为 对照 （Driver mRNA)。 具体歩骤如下：  The SSH library (subtractive library) was constructed by inhibition subtractive hybridization according to the method described in Clontech's PCR-selectTM cDNA Subtraction Kit kit. The mRNA of the leaves of the drought-treated cotton seedlings was used as a sample (Tester mRNA) during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a control (Driver mRNA). 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液体培养基（含 9.39 mM KN03， 0.625 mM KH2P04， 10.3 mM NH4N03 , 0.75 mM MgSO4， 1.5 mM CaCl2， 50 μΜ ΚΙ, 100 μΜ Η3ΒΟ3， 100 μΜ MnS04， 30 μΜ ZnS04, 1 μΜ Να2Μο04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 μΜ FeS04) 一次。 当苗株高达 25-30 cm时用于实验。 冀棉14 (National Cotton Medium-term Repository, obtained by the China Cotton Research Institute, Uniform No.: ZM-30270) Seeded onto sterilized vermiculite at 25 °C, photoperiod 16h light / 8h dark (light intensity 2000-3000) Cultured under conditions of Lx), 1/2 MS liquid medium (containing 9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM NH 4 N0 3 , 0.75 mM MgSO 4 , 1.5 mM CaCl 2 , 50 μΜ ΚΙ, per week). 100 μΜ Η 3 ΒΟ 3 , 100 μΜ MnS0 4 , 30 μΜ ZnS0 4 , 1 μΜ Να 2 Μο0 4 , 0.1 μΜ CoCl 2 , 100 μΜ Na 2 EDTA, 100 μΜ FeS0 4 ) once. It was used for experiments when the seedlings were as high as 25-30 cm.
( 2 ) 材料处理：  (2) Material handling:
将上述供试幼苗分为 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， 止常浇灌。 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 above 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 16 h light / 8 h dark, and was usually 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 two of the seedlings were cut 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各 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 定所得总 RNA在 260 nm和 280 nm的吸光度值， OD260/OD280比值为 1.8-2.0， 表明 总 RNA纯度较高； 用 1.0%的琼脂糖凝胶电泳检测总 RNA的完整性， 28S条带的亮 度约为 18 S条带的 2倍，表明 RN A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 and drought-treated groups were each 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 was measured by HITACHI's UV spectrophotometer U-2001. The ratio of OD 260 / OD 280 was 1.8-2.0, indicating that the total RNA purity was higher; 1.0% agarose gel was used. Gel electrophoresis detects total RNA integrity, bright 28S bands The degree is about 2 times that of the 18 S band, indicating that the integrity of the RN A is good. 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- select™ cDN A Subtraction Kit试剂盒说明书所示的方法进行 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和 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 was performed as indicated by Clontech's PCR-selectTM cDN A Subtraction Kit kit instructions. The Driver mRNA and Tester mRNA were reverse transcribed to obtain a double-stranded cDNA, and then subtracted hybridization using 2 Tester cDNA and 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，除 Rsal酶以外，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, except for the Rsal enzyme, this experiment uses the endonuclease Haelll The above steps were carried out by digestion of the Tester cDNA and the Driver cDNA, followed by two positive subtractive hybridizations and two inhibitory PCR amplifications, and finally the second inhibitory PCR products of the two sets of forward subtractive hybridization cDNA fragments were combined.
( 5 ) 差减文库的构建与初步筛选、 克隆及鉴定  (5) Construction and preliminary screening, cloning and identification of subtractive libraries
依照 pGEM-T Easy试剂盒 （购自 Promega) 的产品说明书所示方法， 将上述合并的 正向差减杂交 cDNA片段的第二次 PCR产物 （使用 QIAquick PCR Purification Kit纯化， 购自 Qiagen)与 pGEM-T Easy载体连接， 其具体步骤如下： 向 200 μΐ PCR管中依次加入 下列成分：纯化的合并后的正向差减杂交 cDNA片段的第二次抑制性 PCR产物 3 μ1, 2χΤ4 DNA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 μ 连接反应产物，加入到 100 μ 感受态大肠杆菌 JM109 (购自 TAKARA) 中 并混匀，冰浴 30 min、42°C热休克 60 s、冰浴 2 min,另加 250 μL L 培养液（含 1% Tryptone 购自 OXOID, 0.5% Yeast Extract购自 OXOID, 1% NaCl购自国药）后置于 37°C摇床中， 以 225 r/min振荡培养 30 min,所得菌液即为差减文库菌液。加甘油至终浓度 20% (V/V) , 于 -80°C保存备用。  The second PCR product of the above combined positive subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) and pGEM according to the method described in the product specification of pGEM-T Easy kit (purchased from Promega) -T Easy vector ligation, the specific steps are as follows: The following components are sequentially added to a 200 μΐ PCR tube: the second inhibitory PCR product of the purified combined positive subtractive hybridization cDNA fragment 3 μ1, 2χΤ4 DNA ligase buffer 5 μl of liquid, pGEM-T Easy vector 1 μl, T4 DNA ligase 1 μΐ, and ligated overnight at 4 °C. 10 μ l of the reaction product was added to 100 μ of competent E. coli JM109 (purchased from TAKARA) and mixed, ice bath for 30 min, heat shock at 42 ° C for 60 s, ice bath for 2 min, plus 250 μL L culture. The solution (containing 1% Tryptone from OXOID, 0.5% Yeast Extract from OXOID, 1% NaCl purchased from Sinopharm) was placed in a 37 ° C shaker and shaken at 225 r/min for 30 min. Subtract the library bacteria solution. Glycerin was added to a final concentration of 20% (V/V) and stored at -80 °C until use.
取 200 所述差减文库菌液涂布于含 50 g/mL氨苄青霉素 （购自北京拜尔迪）、 40 g/mL X-gal ( 5-溴 -4氯 -3-吲哚 - β -D-半乳糖苷）、 24 g/mL IPTG (异丙基 - β -D-硫代吡喃 半乳糖苷） （X-gal和 IPTG均购自 TAKARA) 的 LB (同上） 固体培养平板上， 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 198个白色菌落 (编 号： Gh-B001至 Gh-B198)。 将所有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-select™ cDNA Subtraction Kit试剂盒自带）进行菌液 PCR扩增验证， 得到 190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。 Taking 200 of the subtractive library solution was applied to 50 g/mL ampicillin (purchased from Beijing Bayerdi), 40 g/mL X-gal (5-bromo-4-chloro-3-indol-β- D-galactoside), 24 g/mL IPTG (isopropyl-β-D-thiogalactopyranoside) (X-gal and IPTG are purchased from TAKARA) on LB (ibid.) solid culture plates, Incubate at 37 ° C for 18 h. Count the number of clear white and blue colonies > 1 mm in diameter in the culture plate and randomly pick 198 white colonies No.: Gh-B001 to Gh-B198). All white colonies 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. The nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) were used to verify the PCR amplification of the bacteria, and 190 positive clones were obtained, and then all the positive clones were sent to the UK. Base (Shanghai) Trading Co., Ltd. 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 135 条 EST(Unigene；)。 经分析有 21个重叠群， 有 114个单一的序列。 经 BlastN发现其中 48条 EST(Unigene) 在 GenBank 中有同源序列， 32条 EST功能未知或者为假定蛋白， 另有 34 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 棉花亮氨酸拉链蛋白编码基因 G )Z/P-3的克隆  After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 135 ESTs (Unigene;) were obtained. After analysis, there were 21 contigs with 114 single sequences. It was found by BlastN that 48 ESTs (Unigene) have homologous sequences in GenBank, 32 ESTs are unknown or hypothetical proteins, and another 34 have not obtained homologous matches, presumably at the 3' or 5' end. A shorter sequence of regions. Example 2 Cloning of a cotton leucine zipper protein encoding gene G)Z/P-3
将编号为 Gh-B98的阳性克隆的测序结果去掉冗余 DNA后，序列为 SEQ ID No: 3， 序列分析表明该序列编码的蛋白质属于亮氨酸拉链蛋白， 本文将 SEQ ID No: 3对应的 全长编码基因命名为 GhbZIP-3， 其对应的蛋白命名为 bZIP-3。  After the sequencing result of the positive clone numbered Gh-B98 was removed from the redundant DNA, the sequence was SEQ ID No: 3. The sequence analysis indicated that the protein encoded by the sequence belonged to the leucine zipper protein, and the SEQ ID No: 3 corresponding to the SEQ ID No: 3 The full-length coding gene was named GhbZIP-3, and its corresponding protein was named bZIP-3.
SEQ ID No: 3 SEQ ID No: 3
1 GGTGGTAATC AAAATGGTAT TCAAGCTTCC GATTTGCCTA TGAATTTTAA TGGAGTTAGA 1 GGTGGTAATC AAAATGGTAT TCAAGCTTCC GATTTGCCTA TGAATTTTAA TGGAGTTAGA
61 TCCAATCAGC ACCACTTAAC TCAGCTTCAG CAGCAACCAT TATGTCCTAA GCAACCTGGT61 TCCAATCAGC ACCACTTAAC TCAGCTTCAG CAGCAACCAT TATGTCCTAA GCAACCTGGT
12 1 GTGGAATATG GAGCTCAGAT GGGTTTGCAA AGTGGTGGTC AGTTGGGGAG TCCTGGAATT12 1 GTGGAATATG GAGCTCAGAT GGGTTTGCAA AGTGGTGGTC AGTTGGGGAG TCCTGGAATT
18 1 AGGTGTGGTC AGGGACTATA TAATGGCTTG ATCCATGGTG GTGGGATGAG TATGGTTGGT18 1 AGGTGTGGTC AGGGACTATA TAATGGCTTG ATCCATGGTG GTGGGATGAG TATGGTTGGT
24 1 TTAAGATCAC CTGCTAACCA CTTTTCATCA GATGGGATTG GGAAGAGTAG TGGAGATAGT24 1 TTAAGATCAC CTGCTAACCA CTTTTCATCA GATGGGATTG GGAAGAGTAG TGGAGATAGT
3 0 1 TCCTCAGTTT CACCCGTTCC TTATGTGTTT AATGGAAGTT TGAGGGGTAG GAAAAGCAGT3 0 1 TCCTCAGTTT CACCCGTTCC TTATGTGTTT AATGGAAGTT TGAGGGGTAG GAAAAGCAGT
36 1 GCTGTGGAAA AGGTTGCTGA GAGGAGGCAG AGAAGAATGA TAAAGAACCG AGAATCCGCT36 1 GCTGTGGAAA AGGTTGCTGA GAGGAGGCAG AGAAGAATGA TAAAGAACCG AGAATCCGCT
42 1 GCAAGATCAC GAGCTCGCAA GCAGGCTTAT ACAACGGAGT TGGAAGCAGA AGTTGCTAAG42 1 GCAAGATCAC GAGCTCGCAA GCAGGCTTAT ACAACGGAGT TGGAAGCAGA AGTTGCTAAG
4 8 1 CTGAAAGAGG AGAACCAAGA ATTGCAGAAG AAACA GA C ACATCATAAA AATACAGAAA4 8 1 CTGAAAGAGG AGAACCAAGA ATTGCAGAAG AAACA GA C ACATCATAAA AATACAGAAA
54 1 AATCAGGTGA TGGAGATGAT GAATGTGCAG CCAGGAGCTA AGAAACGATG CTTGCGACGA54 1 AATCAGGTGA TGGAGATGAT GAATGTGCAG CCAGGAGCTA AGAAACGATG CTTGCGACGA
60 1 ACACGGACAG GTCCTTGGTG AATC 60 1 ACACGGACAG GTCCTTGGTG AATC
GhbZIP-3全长编码基因的克隆 Cloning of the full-length coding gene of GhbZIP-3
根据已经获得的 SEQ ID No: 3序列分析: SEQ ID No: 3为编码基因 的 According to the sequence analysis of SEQ ID No: 3 which has been obtained: SEQ ID No: 3 is the coding gene
3 '端序列。 根据已经获得的 SEQ ID NO: 3序列， 设计如下三条特异性引物， 作为反转录引 物及 5 'RACE的 3 '端特异性引物。 3 'end sequence. Based on the sequence of SEQ ID NO: 3 that has been obtained, the following three specific primers are designed as reverse transcription primers. And 5 'end specific primers for 5 'RACE.
GhB98 GSP1 : SEQ ID NO: 4： GhB98 GSP1 : SEQ ID NO: 4:
GACCACCACTTTGCAAACCC  GACCACCACTTTGCAAACCC
GhB98 GSP2 : SEQ ID NO: 5：  GhB98 GSP2 : SEQ ID NO: 5:
CATATTCCACACCAGGTTGC  CATATTCCACACCAGGTTGC
GhB98 GSP3 : SEQ ID NO:6:  GhB98 GSP3 : SEQ ID NO:6:
GGTGGTAATCAAAATGGTATTC  GGTGGTAATCAAAATGGTATTC
试剂盒自带通用引物：  The kit comes with universal primers:
AAP: SEQ ID NO: 7：  AAP: SEQ ID NO: 7:
GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP : SEQ ID NO: 8：  GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP : SEQ ID NO: 8:
GGCCACGCGTCGACTAGTAC 实验步骤按试剂盒说明书操作 （5' RACE System for Rapid Amplification of cDNA Ends试剂盒购自 Invitrogen公司）。  The GGCCACGCGTCGACTAGTAC experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
以 GhB98 GSP1(SEQ ID NO: 4)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: 4与通用引物 SEQ ID NO: 7 (试剂盒自带， I 为次黄嘌吟修饰的 a、 c、 g或 t)， 具体步骤如下：  GhB98 GSP1 (SEQ ID NO: 4) was used as a reverse transcription primer, and reverse transcription was performed using cotton mRNA as a template to obtain a cDNA template. Then, according to the procedure in the above 5' RACE kit instructions, Poly C tail was added to end the tail. The latter product was subjected to the first round of PCR amplification using the primers of SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (the kit is self-contained, and I is hypoxanthine modified a, c, g or t ), 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 并加 Poly C尾后的 cDNA、 1.0 μΐ Ex Taq (购自 TAKARA)、 10 μΜ的引物 SEQ ID NO: 4 禾口 SEQ ID NO: 7各 2.0 μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33 个循环 （ 94 变性 30 s， 56°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 transcription and Poly C tail cDNA, 1.0 μΐ Ex Taq (purchased from TAKARA), 10 μΜ primer SEQ ID NO: 4 and SEQ ID NO: 7 each of 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (94 denaturation for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
将所得的 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：  The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 5 and the general primer SEQ ID NO: 8, and the specific steps were 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 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round
PCR产物、 1.0 l Ex Taq、 10 μΜ的引物 SEQ ID NO: 5和 SEQ ID NO: 8各 2.0 μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 （ 94 变性 45 s， 56 °C 退火 45 s， 72V 延伸 1 min)， 72 °C 延伸 10 min。 回收第二轮 PCR产物中约为 750 bp 大小的条带 （ Gel Extraction Kit购自 OMEGA)， 并将其连接到 pGEM-T Easy载体， 然后 转化到大肠杆菌 JM109 感受态细胞中（具体方法同上)，并将转化后的菌液涂布于含 50 μ§/ηΛ氨苄青霉素的 LB固体培养基上进行筛选。 随机挑取 10个白色菌落分别接种于含 有 50 g/mL氨苄青霉素的 LB 液体培养基中培养， 37°C培养过夜后加甘油至终浓度 20%， -80°C保存备用。用引物 SEQ ID NO: 5与 3'端引物 SEQ ID NO: 6进行菌液 PCR扩增（反 应体系及反应条件同上）， 得到 3个阳性克隆（Gh9-1、 Gh9-5和 Gh9-8)，送英潍捷基（上 海） 贸易有限公司测序，获得该基因的 cDNA的一段 5'端序列。 PCR product, 1.0 l Ex Taq, 10 μΜ primers SEQ ID NO: 5 and SEQ ID NO: 8 each 2.0 μl, and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (94 denaturation for 45 s, annealing at 56 °C for 45 s, 72 V for 1 min), extension at 72 °C for 10 min. A band of about 750 bp in size from the second round of PCR product (GEL extraction kit from OMEGA) was recovered and ligated into pGEM-T Easy vector, which was then transformed into E. coli JM109 competent cells (see above) And coating the transformed bacterial solution in 50 Screening was performed on LB solid medium of μ § /η ampicillin. Ten white colonies were randomly picked and inoculated 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. The primers SEQ ID NO: 5 and the 3' primer SEQ ID NO: 6 were used for PCR amplification (reaction system and reaction conditions as above), and three positive clones (Gh9-1, Gh9-5 and Gh9-8) were obtained. , sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. to sequence, obtain a 5' end sequence of the cDNA of the gene.
将所得的 5'RACE产物中阳性克隆 Gh9-8测序获得序列为 SEQ ID NO: 9： The positive clone Gh9-8 in the obtained 5' RACE product was sequenced to obtain the sequence of SEQ ID NO: 9:
1 GGGGGGGGCA CATGAACTTC AAGAACTTTG GAAGTAACCA ACCGCCGTCA GGTGATGGCG1 GGGGGGGGCA CATGAACTTC AAGAACTTTG GAAGTAACCA ACCGCCGTCA GGTGATGGCG
61 GCGGTGGTAA TAAACCGCCT GGGAATTATC CGATAAGTAG ACAGCCATCG GTCTATTCTC61 GCGGTGGTAA TAAACCGCCT GGGAATTATC CGATAAGTAG ACAGCCATCG GTCTATTCTC
12 1 TAACCTTTGA TGAGTTTCAA AGCACAATGG GTGGAATAGG CAAAGATTTC GGGTCGATGA12 1 TAACCTTTGA TGAGTTTCAA AGCACAATGG GTGGAATAGG CAAAGATTTC GGGTCGATGA
18 1 ACATGGATGA GCTGTTAAAG AACATTTGGA GTGCTGAAGA AACTCAAACA ATGGCTTCTC18 1 ACATGGATGA GCTGTTAAAG AACATTTGGA GTGCTGAAGA AACTCAAACA ATGGCTTCTC
24 1 CCGGTGTTGG CCTAGTGGGA AATGGAGGGT ACAAAGGCA AGGGTCTTTG ACTTTGCCCA24 1 CCGGTGTTGG CCTAGTGGGA AATGGAGGGT ACAAAGGCA AGGGTCTTTG ACTTTGCCCA
3 0 1 GGACACTTAG CCACAAAACT GTTGATGAAG TTTGGAGAGA GA CTCAAAT GAGTTTTCTA3 0 1 GGACACTTAG CCACAAAACT GTTGATGAAG TTTGGAGAGA GA CTCAAAT GAGTTTTCTA
36 1 TAGGGACTGA AGCCACTGAT AATATGCCAC AAAGACAGCA AACCTTAAAG GAGATTACTT36 1 TAGGGACTGA AGCCACTGAT AATATGCCAC AAAGACAGCA AACCTTAAAG GAGATTACTT
42 1 TAGAGGAGTT TTTGGTCAGA GCCGGTGTGG TAAGAGAGGA TAACATCCAA TTTTCTGGGA42 1 TAGAGGAGTT TTTGGTCAGA GCCGGTGTGG TAAGAGAGGA TAACATCCAA TTTTCTGGGA
4 8 1 AGGACAATAA TGTTAATGGC GGCTTCTTTG GGGAATTACC GCAAGCAGGG AGTAA ACCA4 8 1 AGGACAATAA TGTTAATGGC GGCTTCTTTG GGGAATTACC GCAAGCAGGG AGTAA ACCA
54 1 GTGGGTTTGG GATTGGGTTT CAACAAGGTG GAAGAGGTCC AAATTTGATG GGGAATAGGC54 1 GTGGGTTTGG GATTGGGTTT CAACAAGGTG GAAGAGGTCC AAATTTGATG GGGAATAGGC
60 1 TACCTGATGG TGGTAATCAA AATGGTATTC AAGCTTCCGA TTTGCCTATG AATTTTAATG60 1 TACCTGATGG TGGTAATCAA AATGGTATTC AAGCTTCCGA TTTGCCTATG AATTTTAATG
66 1 GAGTTAGATC CAATCAGCAC CACTTAACTC AGCTTCAGCA GCAACCATTA TGTCCTAAGC66 1 GAGTTAGATC CAATCAGCAC CACTTAACTC AGCTTCAGCA GCAACCATTA TGTCCTAAGC
72 1 AACCTGGTGT GGAATATG 将 5 'RACE获得的序列 SEQ ID NO: 9， 与上述获得的序列 SEQ ID NO: 3拼接， 获得72 1 AACCTGGTGT GGAATATG The sequence obtained by 5 'RACE SEQ ID NO: 9, was spliced with the sequence SEQ ID NO: 3 obtained above,
SEQ ID NO: 10： SEQ ID NO: 10:
1 GGGGGGGGCA CATGAACTTC AAGAACTTTG GAAGTAACCA ACCGCCGTCA GGTGATGGCG 1 GGGGGGGGCA CATGAACTTC AAGAACTTTG GAAGTAACCA ACCGCCGTCA GGTGATGGCG
61 GCGGTGGTAA TAAACCGCCT GGGAATTATC CGATAAGTAG ACAGCCATCG GTCTATTCTC61 GCGGTGGTAA TAAACCGCCT GGGAATTATC CGATAAGTAG ACAGCCATCG GTCTATTCTC
12 1 TAACCTTTGA TGAGTTTCAA AGCACAATGG GTGGAATAGG CAAAGATTTC GGGTCGATGA12 1 TAACCTTTGA TGAGTTTCAA AGCACAATGG GTGGAATAGG CAAAGATTTC GGGTCGATGA
18 1 ACATGGATGA GCTGTTAAAG AACATTTGGA GTGCTGAAGA AACTCAAACA ATGGCTTCTC18 1 ACATGGATGA GCTGTTAAAG AACATTTGGA GTGCTGAAGA AACTCAAACA ATGGCTTCTC
24 1 CCGGTGTTGG CCTAGTGGGA AATGGAGGGT TACAAAGGCA AGGGTCTTTG ACTTTGCCCA24 1 CCGGTGTTGG CCTAGTGGGA AATGGAGGGT TACAAAGGCA AGGGTCTTTG ACTTTGCCCA
3 0 1 GGACACTTAG CCACAAAACT GTTGATGAAG TTTGGAGAGA GATCTCAAAT GAGTTTTCTA3 0 1 GGACACTTAG CCACAAAACT GTTGATGAAG TTTGGAGAGA GATCTCAAAT GAGTTTTCTA
36 1 TAGGGACTGA AGCCACTGAT AATATGCCAC AAAGACAGCA AACCTTAAAG GAGATTACTT36 1 TAGGGACTGA AGCCACTGAT AATATGCCAC AAAGACAGCA AACCTTAAAG GAGATTACTT
42 1 TAGAGGAGTT TTTGGTCAGA GCCGGTGTGG TAAGAGAGGA TAACATCCAA TTTTCTGGGA42 1 TAGAGGAGTT TTTGGTCAGA GCCGGTGTGG TAAGAGAGGA TAACATCCAA TTTTCTGGGA
4 8 1 AGGACAATAA TGTTAATGGC GGCTTCTTTG GGGAATTACC GCAAGCAGGG AGTAATACCA4 8 1 AGGACAATAA TGTTAATGGC GGCTTCTTTG GGGAATTACC GCAAGCAGGG AGTAATACCA
54 1 GTGGGTTTGG GATTGGGTTT CAACAAGGTG GAAGAGGTCC AAATTTGATG GGGAATAGGC54 1 GTGGGTTTGG GATTGGGTTT CAACAAGGTG GAAGAGGTCC AAATTTGATG GGGAATAGGC
60 1 TACCTGATGG TGGTAATCAA AATGGTATTC AAGCTTCCGA TTTGCCTATG AATTTTAATG60 1 TACCTGATGG TGGTAATCAA AATGGTATTC AAGCTTCCGA TTTGCCTATG AATTTTAATG
66 1 GAGTTAGATC CAATCAGCAC CACTTAACTC AGCTTCAGCA GCAACCATTA TGTCCTAAGC66 1 GAGTTAGATC CAATCAGCAC CACTTAACTC AGCTTCAGCA GCAACCATTA TGTCCTAAGC
72 1 AACCTGGTGT GGAATATGGA GCTCAGATGG GTTTGCAAAG TGGTGGTCAG TTGGGGAGTC72 1 AACCTGGTGT GGAATATGGA GCTCAGATGG GTTTGCAAAG TGGTGGTCAG TTGGGGAGTC
78 1 CTGGAATTAG GTGTGGTCAG GGACTATATA ATGGCTTGAT CCATGGTGGT GGGATGAGTA78 1 CTGGAATTAG GTGTGGTCAG GGACTATATA ATGGCTTGAT CCATGGTGGT GGGATGAGTA
84 1 TGGTTGGTTT AAGATCACCT GCTAACCACT TTTCATCAGA TGGGATTGGG AAGAGTAGTG 90 1 GAGATAGTTC CTCAGTTTCA CCCGTTCCTT ATGTGTTTAA TGGAAGTTTG AGGGGTAGGA84 1 TGGTTGGTTT AAGATCACCT GCTAACCACT TTTCATCAGA TGGGATTGGG AAGAGTAGTG 90 1 GAGATAGTTC CTCAGTTTCA CCCGTTCCTT ATGTGTTTAA TGGAAGTTTG AGGGGTAGGA
96 1 AAAGCAGTGC TGTGGAAAAG GTTGCTGAGA GGAGGCAGAG AAGAATGATA AAGAACCGAG96 1 AAAGCAGTGC TGTGGAAAAG GTTGCTGAGA GGAGGCAGAG AAGAATGATA AAGAACCGAG
1021 AATCCGCTGC AAGATCACGA GCTCGCAAGC AGGCTTATAC AACGGAGTTG GAAGCAGAAG1021 AATCCGCTGC AAGATCACGA GCTCGCAAGC AGGCTTATAC AACGGAGTTG GAAGCAGAAG
10 81 TTGCTAAGCT GAAAGAGGAG AACCAAGAAT TGCAGAAGAA ACATGATCAC A CA AAAAA10 81 TTGCTAAGCT GAAAGAGGAG AACCAAGAAT TGCAGAAGAA ACATGATCAC A CA AAAAA
1141 TACAGAAAAA TCAGGTGATG GAGATGATGA ATGTGCAGCC AGGAGCTAAG AAACGATGCT1141 TACAGAAAAA TCAGGTGATG GAGATGATGA ATGTGCAGCC AGGAGCTAAG AAACGATGCT
12 01 TGCGACGAAC ACGGACAGGT CCTTGGTGAA C 12 01 TGCGACGAAC ACGGACAGGT CCTTGGTGAA C
根据 SEQ ID NO: 10序列设计一对引物如下：  A pair of primers were designed according to the sequence of SEQ ID NO: 10 as follows:
GhbZIP-3F: SEQ ID NO: 11： GhbZIP-3F: SEQ ID NO: 11:
ATGAACTTCAAGAACTTTGGA ATGAACTTCAAGAACTTTGGA
GhbZIP-3R: SEQ ID NO: 12： GhbZIP-3R : SEQ ID NO: 12:
TCACCAAGGACCTGTCCGT AP: SEQ ID NO: 13： TCACCAAGGACCTGTCCGT AP: SEQ ID NO: 13:
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 通过 SEQ ID NO: 11和 SEQ ID NO: 12来克隆 GhbZIP-3全长编码序列。  GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT The GhbZIP-3 full-length coding sequence was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
提取棉花 RNA,以引物 SEQ ID NO: 13 为反转录引物，获取棉花 cDNA。 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: 11和 SEQ ID NO: 12各 2.0 μ1，以及 37.5 μΐ的双蒸水。 PCR反应条件： 95 °C预变性 2 min， 35个循环 （95 °C 变性 25 s， 53 °C退火 30 s， 72 °C 延伸 1 min)， 72 °C 延伸 5 min。  Cotton RNA was extracted and primer cDNA SEQ ID NO: 13 was used as a reverse transcription primer to obtain cotton cDNA. Stratagene's PfuUltra II Fusion HS DNA Polymerase was used for PCR reaction 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: 11 and SEQ ID NO: 12 each 2.0 μ1 , 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 53 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 5 min.
PCR扩增产物加 A尾： PCR产物加 2.5倍体积的无水乙醇， -20°C放置 10 min, 离心， 去上清， 晾干， 用 21 μΐ双蒸水溶解。 加入 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μΐ 5 mM的 dATP ， 1.0 l Ex Taq。 反应条件： 70°C反应 30 min。 将得到的约 1200bp的 DNA片 段回收 （Omega回收试剂盒） ， 并将其连接至 pGEM T-easy载体上 （得到  PCR amplification product plus A tail: PCR product plus 2.5 volumes of absolute ethanol, placed at -20 ° C for 10 min, centrifuged, supernatant, dried, dissolved in 21 μl double distilled water. Add 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μΐ 5 mM dATP, 1.0 l Ex Taq. Reaction conditions: 70 ° C reaction for 30 min. The obtained 1200 bp DNA fragment was recovered (Omega Recovery Kit) and ligated to the pGEM T-easy vector (obtained)
GhbZIP-3 -pGEM质粒） ，然后转化到大肠杆菌 JM109感受态细胞中并在含 50 g/mL 氨苄青霉素的 LB固体培养基上进行筛选 （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: 1 1与 SEQ ID NO: 12进行菌液 PCR 扩增 （反应体系及反应条件同上）， 得到 3个阳性克隆，送至英潍捷基 （上海） 贸易有 限公司测序，所得序列为 SEQ ID NO: 2，其编码的蛋白的氨基酸序列为 SEQ ID NO: 1。 ¥D¥¥OD¥ODO iiDOi¥OD¥¥ ¥0¥¥iDO¥00 ¥DDO¥DOiOi ¥¥0i¥0i¥0¥ OOi¥OiOO¥D T^TT i¥¥¥¥¥0¥D¥ i¥¥¥¥¥i¥Di ¥D¥Di¥Oi¥D ¥¥¥0¥¥0¥D0 ii¥¥0¥¥DD¥ ¥0¥00¥0¥¥¥ T80TGhbZIP-3 -pGEM plasmid) was then transformed into E. coli JM109 competent cells and screened on LB solid medium containing 50 g/mL ampicillin (method supra). Eight white colonies were randomly picked and inoculated 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. The primers SEQ ID NO: 1 1 and SEQ ID NO: 12 were used for PCR amplification (the reaction system and the reaction conditions are the same as above), and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The sequence is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is SEQ ID NO: 1. ¥D¥¥OD¥ODO iiDOi¥OD¥¥ ¥0¥¥iDO¥00 ¥DDO¥DOiOi ¥¥0i¥0i¥0¥ OOi¥OiOO¥DT^TT i¥¥¥¥¥0¥D¥ i¥ ¥¥¥¥i¥Di ¥D¥Di¥Oi¥D ¥¥¥0¥¥0¥D0 ii¥¥0¥¥DD¥ ¥0¥00¥0¥¥¥ T80T
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ZL 69linOZ OAV 12 01 CGGACAGGTC CTTGGTGA 实施例 3 GhbZIP-3基因植物表达载体构建 ZL 69linOZ OAV 12 01 CGGACAGGTC CTTGGTGA Example 3 Construction of GhbZIP-3 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 ΡΤΠ蛋白在植物中的表达。 选择含双增强子的 35S启动子及 Tnos终止子分 别作为 G )Z/P-3基因的启动子和终止子， 构建流程如图 1所示。  The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the 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 Tnos terminator were selected as promoters and terminators of the G)Z/P-3 gene, respectively. The construction process is shown in Figure 1.
使用引物 SEQ ID NO: M SEQ ID NO: 15以植物表达载体 pBI121 (购自北京华 夏远洋科技有限公司）为模板扩增 Pnos，采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer、 3 μΐ 2.5 mM的 dNTP、 1.0 μΐ pBI121 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 14和 SEQ ID NO: 15各 2.0 μ1， 以及 31 μΐ的双 蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环（94 °C 变性 30 s， 56°C退火 30 s， 72 °C 延伸 30 s)， 72 °C 延伸 10 min。 通过 EcoRI、 Bglll酶切后将所得 PCR产物连接 至 lj pCAMBIA2300 (Promega, T4连接酶盒)获得 pCAMBIA2300-l。  Primer SEQ ID NO: M SEQ ID NO: 15 was used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 14 and SEQ ID NO: 15 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. The resulting PCR product was digested with EcoRI and Bglll and ligated into lj pCAMBIA2300 (Promega, T4 ligase cassette) to obtain pCAMBIA2300-1.
SEQ ID NO : 14 ： SEQ ID NO: 14:
GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO : 15：  GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO : 15:
ATCCAGATCTAGATCCGGTGCAGATTATTTG  ATCCAGATCTAGATCCGGTGCAGATTATTTG
使用引物 SEQ ID NO: 16和 SEQ ID NO: 17以 pBI121为模板扩增 Tnos， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 1.0 μΐ pBI121 , 1.0 μΐ Prime STAR、 10 μΜ的引物 SEQ ID NO: 16和 SEQ ID NO: 17各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 (94°C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 30 s)， 72 °C 延伸 10 min。 通过 Sall、 EcoRI酶切后将所得 PCR产物连接到 pCAMBIA2300-1 (Promega T4 连接酶盒) 获得 pCAMBIA2300-2。 SEQ ID NO : 16：  Primers SEQ ID NO: 16 and SEQ ID NO: 17 were used to amplify Tnos using pBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ Prime STAR, 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μ1, 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 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 Sall, EcoRI and ligated into pCAMBIA2300-1 (Promega T4 ligase cassette) to obtain pCAMBIA2300-2. SEQ ID NO : 16:
GC^rC^CGAATTTCCCCGATCGTTCAAA  GC^rC^CGAATTTCCCCGATCGTTCAAA
SEQ ID NO : 17： SEQ ID NO: 17:
CAGAA rmXAGTGAATTCCCGATCTAGTA  CAGAA rmXAGTGAATTCCCGATCTAGTA
使用引物 SEQ ID NO: 18和 SEQ ID NO: 19以 pCAMBIA2300质粒为模板扩增 35S 启动子。采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 l 5 ><PS Buffer 3 μΐ 2.5 mM的 dNTP、1.0 μΐ稀释 50倍的 pCAMBIA2300质粒、 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO:18禾 P SEQ ID NO:19各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR 反应条件： 94°C预变性 5 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 The 35S promoter was amplified using the primers SEQ ID NO: 18 and SEQ ID NO: 19 using the pCAMBIA2300 plasmid as a template. PrimeSTAR HS DNA polymerase using TaKaRa. 50 μΐ PCR reaction system: 10 l 5 ><PS Buffer 3 μΐ 2.5 mM dNTPs, 1.0 μΐ diluted 50-fold pCAMBIA2300 plasmid, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 18 and P SEQ ID NO: 19 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 50 ° C for 30 s, extension at 72 ° C for 30 s), extension at 72 ° C for 10 min. The resulting PCR product was ligated by HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3
SEQ ID NO: 18： SEQ ID NO: 18:
ACT^UrATGGTGGAGCACGACACTCT  ACT^UrATGGTGGAGCACGACACTCT
SEQ ID NO: 19：  SEQ ID NO: 19:
TGACf^ GAGATAGATTTGTAGAGAGAGAC  TGACf^ GAGATAGATTTGTAGAGAGAGAC
以 SEQ ID NO: 20和 SEQ ID NO: 21为引物， 以实施例 2中含有全长 GhbZIP-3基 因的阳性 GMZH^-pGEM质粒为模板， 扩增两端带有酶切位点的 GMZH^基因。 采 用 stratagene的 PfuUltra II Fusion HS DNA Polymerase。 50 μΐ PCR反应体系： 5 μΐ lO PfuUltra II reaction Buffer 0.5μ125 mM的 dNTP、 2.0 μΐ GhbZIP-3-pGEM质粒、 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ的引物 SEQ ID NO:20和 SEQ ID NO: 21各 2.0 μ1，以及 37.5 μΐ的双蒸水。 PCR反应条件： 95°C预变性 2min， 35个循环（95°C 变性 25 s， 58°C退火 30 s， 72 °C 延伸 45 s)， 72 °C 延伸 5 min。 通过 Pstl、 Sail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-GhbZIP-3-2300 (图 2) 。  Using SEQ ID NO: 20 and SEQ ID NO: 21 as primers, the GMZH^-pGEM plasmid containing the full-length GhbZIP-3 gene in Example 2 was used as a template to amplify GMZH^ with restriction sites at both ends. gene. Stratagene's PfuUltra II Fusion HS DNA Polymerase was used. 50 μΐ PCR reaction system: 5 μΐ lO PfuUltra II reaction Buffer 0.5μ125 mM dNTP, 2.0 μΐ GhbZIP-3-pGEM plasmid, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ primers SEQ ID NO: 20 and SEQ ID NO: 21 each of 2.0 μl, and 37.5 μΐ of double distilled water. PCR reaction conditions: Pre-denaturation at 95 °C for 2 min, 35 cycles (denaturation at 95 °C for 25 s, annealing at 58 °C for 30 s, extension at 72 °C for 45 s), extension at 72 °C for 5 min. After the Pstl and Sail were digested, the obtained PCR product was ligated (the ligation method was the same as above) to pCAMBIA2300-3, and the plant expression vector 35S-GhbZIP-3-2300 was obtained (Fig. 2).
SEQ ID NO: 20： SEQ ID NO: 20:
AAd ATGGGAGATAGATTCATGATG  AAd ATGGGAGATAGATTCATGATG
SEQ ID NO: 21：  SEQ ID NO: 21:
GC^G^CCATAAATGATTTCGCATACAC  GC^G^CCATAAATGATTTCGCATACAC
实施例 4 35S-GMZ/ -3-2300表达载体转化农杆菌  Example 4 Transformation of Agrobacterium by 35S-GMZ/-3-2300 Expression Vector
农杆菌 LBA4404 (购自 Biovector Science Lab,Inc) 感受态细胞的制备： 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 V 培养 1至 2天。 挑取单菌落接种于 5 ml含 50 μ§/ιη1利福平和 50 μ§/ιη1链霉素的 LB液体 培养基中， 28°C下摇动培养过夜 (约 12-16 h)至 OD6(K)值为 0.4， 形成种子菌液。 取 5 ml 活化后的种了菌液（1:20的比例）接种于 100 ml含 50 μ§/ηι1利福平和 50 μ§/ηι1链霉素的 LB液体培养基中， 28°C摇动培养 2-2.5 h至 OD6QQ=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， 即制得 LBA4404 感受态细胞， 以 40 μΐ/管将其分装， 于 -70°C保存备用。 Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was spotted on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, 28 V is incubated for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 μ § /ιη1 rifampicin and 50 μ § /ιη1 streptomycin, and cultured overnight (about 12-16 h) to OD 6 at 28 °C. The K) value is 0.4, and a seed bacterial liquid is formed. 5 ml of the activated seed solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 μ § /ηι1 rifampicin and 50 μ § /ηι1 streptomycin, and cultured at 28 ° C with shaking. 2-2.5 h to OD 6QQ = 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; The cells were resuspended in ice-cold 10% glycerol, centrifuged at 4000 g for 10 min at 4 °C, and the precipitate was collected; washed repeatedly with ice-cold 10% glycerol 3-4 times; 10% pre-cooled by adding appropriate amount of ice bath Glycerol was resuspended in the bacterial pellet to prepare LBA4404 competent cells, which were dispensed at 40 μΐ/tube and stored at -70 °C until use.
转化农杆菌： 在冰上融化 LBA4404感受态细胞， 向 40 μΐ的所述感受态细胞中加入 1 μΐ实施例 3中所得的阳性 35S-G )Zff-3-2300质粒， 混匀后冰浴约 10 min。 将冰浴后 的所述感受态细胞和所述阳性 35S-G )Z/P-3-2300质粒的混合物用微量移液器转移到冰 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 1ml 28°C预热的 LB培养基。 快速而轻柔的用微量移液器将混合物打匀。 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。 Transformation of Agrobacterium: Melt LBA4404 competent cells on ice, add 1 μΐ of the positive 35S-G)Zff-3-2300 plasmid obtained in Example 3 to 40 μΐ of the competent cells, mix and mix, then ice bath 10 min. The mixture of the competent cells after the ice bath and the positive 35S-G)Z/P-3-2300 plasmid was transferred to an ice-cold electric shock cup with a micropipette, and tapped to bring the suspension to the bottom. , be careful not to have 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 program of MicroPuMUer (purchased from Bio-Rad) was set to "Agr" and the shock was applied once. Immediately remove the electric shock cup and add 1 ml of pre-warmed LB medium at 28 °C. Quickly and gently mix the mixture with a micropipette. 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.
实施例 5 利用农杆菌介导的转化法获得转基因拟南芥  Example 5 Agrobacterium-mediated transformation was used to obtain transgenic Arabidopsis thaliana
待转化植株培养： 将拟南芥种子 （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 1/2MS液体培养基 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 1/2MS 液体培养基。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， 以解除顶端优势， 促使多个次生花序 的同步出现。 当大多数花序约 1一 10 cm高 （剪去第一个花序后 4一 8 d) 时准备浸染。  Plant to be transformed: Arabidopsis seeds (Colombian type, Arabidopsis Bioresource Center from Ohio State University) 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. Before transplanting, 40 ml of 1/2 MS liquid medium was poured per pot. After transplanting, the soil moisture was added to the soil in time. Properly water 1/2 MS liquid medium during growth. 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 to relieve the apical dominance and promote the simultaneous emergence of multiple secondary inflorescences. When most of the inflorescences are about 1 to 10 cm high (4 to 8 days after the first inflorescence is cut), they are ready to be infiltrated.
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 OD6(K)=1.8， 然后 3000 r/min室温离心 15 min, 弃去上清液后用浸染培 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 OD600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 to 200 mL of LB liquid medium containing the above antibiotics at a ratio of 1% to 2%, and the concentration of Agrobacterium was adjusted to OD 6 (K) = 1.8 at a constant temperature of 28 °C. Then centrifuge at 3000 r/min for 15 min at room temperature, discard the supernatant and resuspend the Agrobacterium with the dipping medium (Silwet L-77 containing 5.0% sucrose and 0.05% (500 μΙΤί)) and suspend to an OD 600 of about 0.80. .
花序的浸染： 将上述含农杆菌的浸染培养基加入大口容器中， 每个口径 9 cm的容器 中加入 200— 300 mL所述含农杆菌的浸染培养基用于浸染。 将植株倒置， 使地上组织全 部浸没在农杆菌悬浮液中 3— 5 s, 并要轻轻搅动。 浸润后植株上应该有一层液体膜。 浸 染过的植株放在塑料盘中， 用干净的塑料或保鲜膜覆盖以保湿， 然后放置在弱光或暗处 过夜， 注意小心防止阳光直射植株。 处理后约 12— 24 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 T1代转基因种子， 并将所述种子用离心 管在 4 °C下干燥贮存。 Infiltration of inflorescences: Adding the above-mentioned Agrobacterium-containing dyeing medium to a large-mouth container, each container having a diameter of 9 cm 200-300 mL of the Agrobacterium-containing dyeing medium was added for dip dyeing. Invert the plants so that the aboveground tissues are completely immersed in the Agrobacterium suspension for 3-5 s and gently agitated. 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. T1 generation transgenic seeds were harvested and stored in a centrifuge tube at 4 °C.
转基因种子筛选:配制 1/2MS液体培养基，加入 0.8 % (W/V) 琼脂粉，用微波炉加热 至琼脂完全溶化，待冷却到 50°C 左右，加入卡那霉素至终浓度为 50 mg-L"1,摇匀后每培养 皿中倒入 25 mL,置实验台冷却凝固后所得固体培养基即可用于播种。将称量好的种子倒 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 V、 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 1/2MS液体培养基。取生长 20天的卡那霉素抗性拟南芥叶片 0.1 g， 提取 DNA，用 SEQ ID NO: 11和 SEQ ID NO: 12扩增 GhbZIP-3 ( 50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ DNA, 1.0 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 11和 SEQ ID NO: 12 各 2.0 μ1，以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环 （94°C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 1 min)， 72 °C 延伸 7 min)， 将 PCR鉴定为阳性的植株进行编号， 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 T2代转基因种子 （编号为： T2F1-T2F16), 并将种子用离心管在 4 °C下干 燥贮存。 实施例 6 过表达 GhbZIP-3的转基因拟南芥 T2代植株的耐旱模拟实验及功能鉴定 将灭过菌的蛭石用 1/2MS液体培养基浸透。将实施例 5所得的编号为 T2F1-T2F8的 转基因种子及对照拟南芥（非转基因）种子分别播种在所述蛭石上，每盆播种 10颗种子， 25 °C、 10 h光培养 /14 h暗培养循环，每 7天浇一次 1/2MS液体培养基，培养 20天之后， 使用 2000 PPM卡那霉素涂叶片筛选阳性植株，每盆保留大小较一致的 4棵卡那霉素抗性 苗， 用于干旱实验。 转基因拟南芥和对照拟南芥干旱 14天 (不浇水)， 25 °C、 10 h光培养 /14 h暗培养循环。 T2代转基因植株的抗旱性鉴定表明，对照植株都萎蔫严重，而 T2F1、 T2F2 T2F3 T2F4、 T2F5、 T2F6 、 T2F7、 T2F8八个转基因株系共 32棵 （每株系各 4 棵)拟南芥中 30棵能够存活并继续生长显现出显著的耐旱性（参见图 3a和 3b，以 T2F1、 T2F5为例， T2F2 T2F3 T2C 4 T2F6 、 T2F7 T2F8的结果与 T2F1、 T2F5类似， 在 此未示出）。 实施例 7 在转录水平上验证 GhbZIP-3基因的表达 Transgenic seed screening: Prepare 1/2MS liquid medium, add 0.8% (w/v) agar powder, heat in a microwave oven until the agar is completely dissolved, cool to about 50 °C, add kanamycin to a final concentration of 50 mg -L" 1 , pour 25 mL into each dish after shaking, set the solid medium obtained after cooling and solidification on the test bench for seeding. Pour the weighed seeds on a piece of plain copy paper, lightly with your fingers Knock on the copy paper, soak the seeds evenly on the agar gel, cover the Petri dish cover, cool it in a refrigerator at 4 °C for 72 h, then transfer to a 23 V, 16 h light/8 h dark incubator to germination, and periodically count the seed germination. And the growth of the seedlings, the resistant seedlings were transplanted into the nutrient soil in time. After transplanting, the soil moisture was added to the water in time. The 1/2MS liquid medium was properly watered during the growth period. The kanamycin resistance was increased for 20 days. Arabidopsis leaves 0.1 g, DNA extracted, GhbZIP-3 amplified with SEQ ID NO: 11 and SEQ ID NO: 12 (50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ DNA , 1.0 μΐ Ex Taq, 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 2.0 μl, and 35 μΐ 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 1 min), 72 °C extension for 7 min), the plants identified as positive by PCR were numbered, and the plants were cultured normally, and the plants were further grown for 3-5 weeks until the pods became brown and dried. Harvested T2 transgenic seeds (No.: T2F1-T2F16) The seeds were dried and stored in a centrifuge tube at 4 ° C. Example 6 Drought-tolerant simulation experiment and functional identification of transgenic Arabidopsis thaliana T2 plants overexpressing GhbZIP-3 1/2MS of sterilized vermiculite The liquid medium was soaked. The transgenic seeds numbered T2F1-T2F8 obtained in Example 5 and the control Arabidopsis thaliana (non-transgenic) seeds were separately sown on the vermiculite, and 10 seeds were seeded per pot, 25 ° C, 10 h. Light culture/14 h dark culture cycle, 1/2 MS liquid medium was poured every 7 days, after 20 days of culture, positive plants were screened with 2000 PPM kanamycin coated leaves, and 4 Canames with uniform size were retained per pot. Tyrosine resistant seedlings, used in drought experiments. Transgenic Arabidopsis and control Mustard drought for 14 days (without watering), 25 °C, 10 h light culture / 14 h dark culture cycle. The drought resistance of T2 transgenic plants showed that the control plants were wilting, while T2F1, T2F2 T2F3 T2F4, T2F5, Twenty-two transgenic lines of T2F6, T2F7 and T2F8 (4 in each line) 30 of the Arabidopsis thaliana survived and continued to grow with significant drought tolerance (see Figures 3a and 3b, with T2F1). T2F5 is taken as an example. The results of T2F2 T2F3 T2C 4 T2F6 and T2F7 T2F8 are similar to those of T2F1 and T2F5, which are not shown here). Example 7 Validation of GhbZIP-3 Gene Expression at Transcriptional Level
分别取萌发后生长 20天然后干旱 10天的对照拟南芥 （非转基因） 植株、 显著耐旱 转基因拟南芥 T2代植株（分另 'J属于 T2FK T2F2 T2F3 T2F4 T2F5 T2F6六个株系）、 和不显著耐旱转基因拟南芥 T2 代植株的叶片各 0.05 g， 用植物 RNA 提取试剂盒 ( Invitrogen)提取总 RNA。用 HITACHI公司的紫外分光光度计 U-2001测定所得总 RNA 在 260 nm和 280 nm的吸光度值， 计算各个 RNA浓度。 依照 Invitrogen反转录试剂盒 Superscript III Reverse Transcriptase所示方法进行反转录 （2 μg总 RNA作为模板， 反转 录引物 SEQ ID NO： 13 )。通过 SEQ ID NO： 11和 SEQ ID NO: 12扩增 GhbZIP-3，检测 bZIP-3 蛋白相对表达情况。  Control Arabidopsis thaliana (non-transgenic) plants grown after germination for 20 days and then drought for 10 days, and significantly drought-tolerant transgenic Arabidopsis thaliana T2 plants (divided with 'J belongs to T2FK T2F2 T2F3 T2F4 T2F5 T2F6 six strains), The total RNA was extracted with the plant RNA extraction kit (Invitrogen) with 0.05 g of leaves of the drought-tolerant transgenic Arabidopsis thaliana T2 plants. The absorbance values of the total RNA obtained at 260 nm and 280 nm were measured by HITACHI's ultraviolet spectrophotometer U-2001, and the respective RNA concentrations were calculated. Reverse transcription was carried out according to the method shown by Invitrogen Reverse Transcription Kit Superscript III Reverse Transcriptase (2 μg of total RNA as a template, reverse transcription of SEQ ID NO: 13). The relative expression of bZIP-3 protein was examined by amplifying GhbZIP-3 by SEQ ID NO: 11 and SEQ ID NO: 12.
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以上述反转录的 cDNA为模板进 行 PCR反应。 50 μ1 ΡΟ 反应体系： 10 μΐ 5 ><PS Buffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ cDNA、1.0 l PrimeSTAR、10 μΜ的引物 SEQ ID NO: 1 1禾 P SEQ ID NO: 12各 2.0 μ1， 以及 30 μΐ的双蒸水。 PCR反应条件： 94 °C预变性 5 min， 29个循环 （ 94 °C 变性 30 s, 58 °C退火 30 s, 72 V 延伸 45 s ) ， 72 °C 延伸 10 min。  PCR was carried out using TaKaRa's PrimeSTAR HS DNA polymerase using the above reverse transcribed cDNA as a template. 50 μl ΡΟ Reaction system: 10 μΐ 5 ><PS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 l PrimeSTAR, 10 μΜ primer SEQ ID NO: 1 1 and P SEQ ID NO: 12 each 2.0 μl, And 30 μΐ of 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 58 °C for 30 s, extension at 72 V for 45 s), extension at 72 °C for 10 min.
产物电泳结果如图 5所示： M为 DNA Ladder Marker (DL2000,TakaRa) , 1-6为显 著耐旱转基因拟南芥 T2代植株（依次为： T2F1、 T2F2 T2F3 T2F4、 T2F5、 T2F6 )； 7-10 为不显著耐旱转基因拟南芥 T2代植株 ; 11-14为为非转基因拟南芥对照。 图中所示 PCR 产物电泳条带大小与 的大小一致 （约 1200bp ) 。 结果表明， 对照拟南芥没 有 GhbZIP-3转录， 耐旱转基因拟南芥 T2代植株中 GhbZIP-3的转录较强， 不耐旱转 基因拟 ^芥 T2代植株没有转录。  The electrophoresis results of the product are shown in Figure 5: M is DNA Ladder Marker (DL2000, TakaRa), and 1-6 is a significantly drought-tolerant transgenic Arabidopsis thaliana T2 plant (in order: T2F1, T2F2 T2F3 T2F4, T2F5, T2F6); -10 is a T2 generation of transgenic Arabidopsis thaliana plants that are not significantly drought tolerant; 11-14 is a non-transgenic Arabidopsis control. The size of the electrophoresis band of the PCR product shown in the figure is the same as the size (about 1200 bp). The results showed that there was no GhbZIP-3 transcription in Arabidopsis thaliana, and GhbZIP-3 transcription was stronger in T2 generation plants of drought-tolerant transgenic Arabidopsis thaliana plants, and the T2 generation plants of the drought-tolerant transgenic gene were not transcribed.

Claims

权 利 要 求 书 Claim
1. 棉花的一个亮氨酸拉链蛋白， 其序列为 SEQ ID N0: 1。 A leucine zipper protein of cotton having the sequence SEQ ID NO: 1.
2. 编码权利要求 1的亮氨酸拉链蛋白的基因， 其序列为 SEQ ID NO: 2。  2. A gene encoding the leucine zipper protein of claim 1, which has the sequence of SEQ ID NO: 2.
3. 一种重组表达载体， 其是通过将权利要求 2所述的基因插入到一种表达载体 而获得的， 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， 所述表达载体是 pCAMBIA2300。  A recombinant expression vector obtained by inserting the gene of claim 2 into an expression vector, and wherein the nucleotide sequence of the gene and the expression control sequence of the expression vector are operably Preferably, the expression vector is pCAMBIA2300.
4. 权利要求 3所述的载体， 其为附图 2所示的 35S-GMZ/P-3-2300载体。 4. The vector of claim 3 which is the 35S-GMZ/P-3-2300 vector shown in Figure 2.
5. 一种重组细胞， 其含有权利要求 2所述的基因或者权利要求 3或 4所述的重 组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。 A recombinant cell comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4; preferably, the recombinant cell is a recombinant Agrobacterium cell.
6. 一种改善植物耐旱性的方法， 包括： 将权利要求 2所述的基因或者权利要求 3或 4所述的重组表达载体导入植物或植物组织并使所述基因表达； 优选地， 所述植 物是拟南芥。  A method for improving drought tolerance of a plant, comprising: introducing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 into a plant or plant tissue and expressing the gene; preferably, The plant is Arabidopsis.
7. 一种制备转基因植物的方法， 包括： 在有效产生植物的条件下培养含有权利 要求 2所述的基因或者权利要求 3或 4所述的重组表达载体的植物或植物组织。  A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions effective to produce a plant.
8. 权利要求 7所述的方法， 其中所述植物是拟南芥。  8. The method of claim 7, wherein the plant is Arabidopsis thaliana.
9. 权利要求 2所述的基因、 权利要求 3或 4所述的重组表达载体或者权利要求 5所述的重组细胞用于改善植物耐旱性以及用于植物育种的用途。  The gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 5 for use in improving drought tolerance of a plant and for use in plant breeding.
10. 权利要求 9所述的用途， 其中所述植物是拟南芥。  10. The use of claim 9, wherein the plant is Arabidopsis thaliana.
PCT/CN2013/074402 2013-04-19 2013-04-19 Cotton leucine zipper protein bzip-3, coding genes of same, and application thereof WO2014169472A1 (en)

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