WO2013185256A1 - 棉花的一个dreb1类转录因子及其编码基因与应用 - Google Patents

棉花的一个dreb1类转录因子及其编码基因与应用 Download PDF

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WO2013185256A1
WO2013185256A1 PCT/CN2012/000797 CN2012000797W WO2013185256A1 WO 2013185256 A1 WO2013185256 A1 WO 2013185256A1 CN 2012000797 W CN2012000797 W CN 2012000797W WO 2013185256 A1 WO2013185256 A1 WO 2013185256A1
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plant
seq
nucleotide sequence
expression vector
tobacco
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PCT/CN2012/000797
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English (en)
French (fr)
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崔洪志
王建胜
何云蔚
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创世纪转基因技术有限公司
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Priority to CN201280001634.8A priority Critical patent/CN103703020B/zh
Priority to PCT/CN2012/000797 priority patent/WO2013185256A1/zh
Publication of WO2013185256A1 publication Critical patent/WO2013185256A1/zh

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

Definitions

  • the present invention relates to plant transcription factors and coding genes thereof and applications thereof, and in particular to a cotton-derived
  • BACKGROUND OF THE INVENTION Abiotic stresses, such as drought, salting, extreme temperature, chemical pollution and oxygen damage, can cause serious damage to plant growth and development, and cause great loss to crop yield, among which drought has an impact on crop yield. It takes the first place in natural adversity, and its harm is equivalent to the sum of other disasters. It is the bottleneck of agricultural development in many areas. According to statistics, the world's dry and semi-dry areas account for 34% of the land area; China's dry and semi-arid areas account for 52% of the country's land area, and the annual area is 20 to 2.7 million hectares. About 3 billion cubic meters, less than 300-400 million kilograms of food due to lack of water; especially China's main grain-producing areas such as North China, Northeast China and Northwest China are the most severe areas in China, and the spring drought frequently reaches 10 years. .
  • genes and their expression products can be divided into three categories: (1) genes and products involved in signal cascade amplification and transcriptional control; (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) Proteins associated with the uptake and transport of water and ions.
  • genes and products involved in signal cascade amplification and transcriptional control (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) Proteins associated with the uptake and transport of water and ions.
  • proteins associated with the uptake and transport of water and ions Proteins associated with the uptake and transport of water and ions.
  • DREB1 and DREB2 Two transcrip tion facto rs, DREB1 and DREB2, w ith an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought-and low temperature-responsive gene exp Ression, respectively, in A rabidopsis. Plant Cell, 10: 1391-1406; KAN GJY.2002.
  • the present inventors cloned a DNA sequence encoding a DREB (dehydration responsive element binding protein) transcription factor (designated herein as GhCBF2) using a combination of SSH and RACE. It was found that the introduction of the transgenic plants significantly improved the cold resistance and early resistance of the transgenic plants, and these traits were stably inherited.
  • DREB dehydration responsive element binding protein
  • the first aspect of the present invention provides a DREB-like transcription factor GhCBF2 of cotton having the sequence of SEQ ID NO: l o
  • a second aspect of the invention provides a nucleotide sequence encoding the transcription factor of the first aspect of the invention.
  • the nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 2.
  • a third aspect of the invention provides a recombinant expression vector comprising the nucleotide sequence of the second aspect of the invention and the nucleotide sequence is operably linked to an expression control sequence of the expression vector; preferably, The vector is the rd29A-GhCBF2-2300 vector shown in Figure 2.
  • a fourth aspect of the present invention provides a recombinant cell comprising the nucleotide sequence of the second aspect of the present invention or the recombinant expression vector of the third aspect of the present invention; preferably, the recombinant cell is a recombinant Agrobacterium cell .
  • a method for improving cold tolerance and/or drought resistance of a plant according to the fifth aspect of the invention comprising: introducing the nucleotide sequence of the second aspect of the invention or the recombinant expression vector of the third aspect of the invention into a plant or a plant Tissue and expression of the gene; preferably, the plant is tobacco.
  • a method for producing a transgenic plant according to a sixth aspect of the present invention comprising: cultivating a plant comprising the nucleotide sequence of the second aspect of the present invention, the recombinant expression vector of the third aspect of the present invention, under conditions effective for producing a plant Or plant tissue; preferably, the plant is tobacco.
  • a seventh aspect of the present invention provides the transcription factor of the first aspect of the present invention, the nucleoside of the second aspect of the present invention
  • the acid sequence, the recombinant expression vector of the third aspect of the invention or the recombinant cell of the fourth aspect of the invention for improving cold tolerance and/or early resistance of the plant and for use in plant breeding; preferably, said The plant is tobacco.
  • FIG. 1 is a construction flow of a plant expression vector rd29A-GhCBF2-2300) of GhCBF2.
  • Figure 2 is a plasmid map of the plant expression vector (rd29A-GhCBF2-2300) of GhCBF2.
  • FIG. 3 shows the experimental results of cold resistance of transgenic tobacco.
  • the left side of the figure is the transgenic plant (L3-2); the right side is the non-transgenic plant (control).
  • Figure 4 is the experimental results of drought resistance of transgenic tobacco.
  • the left side of the figure is the transgenic plant ( ⁇ 5-11) ; the right side is the non-transgenic plant (control).
  • BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further illustrated by the following non-limiting examples.
  • test seedlings were divided into 2 groups, 4 pots per group and 1 pot per pot.
  • the first group was the control group, cultured at 25 °C
  • the second group was treated with low temperature
  • the leaves of the top 1/3 of the two seedlings were cut out in time, quickly frozen with liquid nitrogen, and stored in a -70 ⁇ refrigerator.
  • the second PCR product of the combined forward subtractive hybridization cDNA fragment (QIAquick PCR Purification Kit purified from Qiagen) was ligated with pGEM-T Easy vector (purchased from Promega) according to the pGEM-T Easy kit product specification.
  • the specific steps are as follows: the following components are sequentially added with a 200 ul PC tube: a second PCR product of the purified cDNA fragment 3 ul, T4 ligase buffer 5 ul, pGEM-T Easy vector l ul, T4 DNA ligase lul, at 4 ⁇ Connect overnight.
  • the reaction product was ligated to ⁇ competent Escherichia coli JM109 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, and 250 ⁇ L LB medium (1% Tryptone was purchased from OXOID, 0.5). % Yeast Extract was purchased from OXOID, 1% NaCl was purchased from Sinopharm.
  • Glycerol was brought to a final concentration of 20% and stored at -80 °C until use.
  • Nested PCR primer Primer 1 (PCR-selectTM cDNA Subtraction Kit kit comes with)
  • Wo D Primer 2R (PCR-select TM cDNA Subtraction Kit kit was included) to carry out PCR amplification of the bacterial liquid, and 152 positive clones were obtained, and all positive clones were sent to Yingjieji (Shanghai) Trading Co., Ltd. for sequencing.
  • G CBF2 GSP1 SEQ ID NO: 4:
  • GhCBF2 GSP2 SEQ ID NO: 5:
  • the experimental procedure was performed according to the kit instructions (3 ' RACE System for Rapid Amplification of cDNA Ends kit, purchased from Invitrogen)
  • the first round of PCR amplification was carried out using SEQ ID NO: 4 and the 3' primer AUAP (provided with the kit), using mRNA reverse transcribed cDNA as a template.
  • the specific steps are as follows: Ex Taq purchased from TAKARA, 50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ Ex Taq, 10 ⁇ primer SEQ ID NO : 4 and AUAP each 2.0 ⁇ 1, and 35 ⁇ double distilled water.
  • PCR reaction conditions 94V Pre-denaturation for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, 33 cycles; extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50-fold with double-distilled water, and 1 ⁇ was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 5 and the 3′ primer AUAP, and the specific steps were as follows: 50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ first round of diluted PCR product, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 5 and AUAP each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; 94V denaturation for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, 33 cycles; extension at 72 °C for 10 min.
  • the second PCR product (QIAquick PCR Purification Kit purified from Qiagen) was ligated to pGEM-T Easy Vector, transformed into E. coli JM109 (specific method as above), and 10 white colonies were randomly picked up to contain 50 ug/mL ampicillin. Penicillin was cultured in LB liquid medium, and cultured overnight at 37 °C, glycerol was added to a final concentration of 20%, and stored at -80 Torr for use.
  • SEQ ID NO: 5 and 3' primer AUAP were used for PCR amplification (system and conditions are the same as above), and 4 positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the cDNA of the gene was obtained. 'end.
  • G CBF2 GSP3 SEQ ID O: 6:
  • GhCBF2 GSP5 SEQ ID O: 8:
  • SEQ ID NO: 6 is a primer that is reverse transcribed into cDNA into mRNA.
  • the first round of PCR amplification was carried out using SEQ ID NO: 7 and 5' universal primer AAP (provided with the kit), and cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 6) was used as a template for the first round of PCR amplification.
  • the specific steps are as follows: Ex Taq was purchased from TAKARA, 50 ⁇ PCR reaction system: 5 ⁇ lOxEx Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ total RNA reverse transcribed cDNA, 1.0 ⁇ ⁇ ⁇ 10 ⁇ primers SEQ ID NO: 7 and AAP Each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 33 cycles; 72 "C extension for 10 min.
  • the obtained PCR product was diluted 50 times with double distilled water, and 1 ⁇ was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 8 and the 3′ primer AUAP, and the specific steps were as follows: 50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ first round of diluted PCR product, 1.0 ⁇ Ex Taq, 10 ⁇ primer SEQ ID NO: 8 and AUAP each 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions predenaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, 33 cycles; extension at 72 °C for 10 min.
  • the second PCR product (QIAquick PCR Purification Kit purified from Qiagen) was ligated to pGEM-T Easy Vector, transformed into JM109 (specific method as above), and 10 white colonies were randomly picked up to contain 50 ug/mL ampicillin. Incubate in LB liquid medium, incubate at 37 °C overnight, add glycerol to a final concentration of 20%, and store at -80 °C until use.
  • SEQ ID NO: 8 and the 3' primer AUAP were used for PCR amplification (reaction system and reaction conditions as above), and 4 positive clones were obtained and sent to Guangzhou Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The 5' end of the cDNA.
  • the obtained 5 ' RACE product was cloned and sequenced, and spliced with the 3 ' RACE product sequencing result.
  • the full-length cDNA sequence of GhCBF2 was obtained.
  • a pair of primers were designed based on the full-length cDNA sequence of GhCBF2 as follows:
  • GhCBF2R SEQ ID NO: 10:
  • the full length of the GhCBF2 encoding gene was cloned by SEQ ID NO: 9 and SEQ ID NO: 10.
  • PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
  • PCR reaction system 10 ⁇ 1 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 1 cDNA, ⁇ . ⁇ ⁇ 1 PrimeSTAR, 10 ⁇ M primer SEQ ID NO: 9 and SEQ ID NO: 10 2.0 ⁇ l, 30 ⁇ each of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 60 s, 33 cycles; extension at 72 °C lOmino
  • PCR amplification product plus A 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.
  • Reaction conditions The reaction was carried out at 70 ° C for 30 minutes.
  • a DNA fragment of about 600 bp was recovered (Omega recovery kit), cloned into pGEM T-easy vector, and transformed into JM109 (method as above). Ten white colonies were randomly picked and cultured in LB liquid medium containing 50 ug/mL ampicillin.
  • SEQ ID NO: 9 and SEQ ID NO: 10 were used for bacterial liquid PCR amplification (reaction system and reaction conditions are the same as above), and 5 positive clones were obtained and sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • the sequence is SEQ ID NO. : 2.
  • Its protein expression sequence is SEQ ID NO: 1.
  • Nucleotide sequence of the GhCBF2 encoding gene SEQ ID NO- 2
  • the plant expression vector rd29A-GhCBF2-2300 was constructed as shown in Figure 1.
  • the plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ⁇ gene was replaced with the Pnos promoter to reduce the prion protein in plants. expression.
  • the inducible promoters rd29A and Tnos were selected as promoters and terminators of the GhCBF2 gene. Specific steps are as follows:
  • Pnos was amplified using the plant expression vector PBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using SEQ ID NO: 11 and SEQ ID NO: 12, 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 ⁇ PrimeSTA, ⁇ ⁇ ⁇ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 ⁇ 1, and 3 1 ⁇ ⁇ double distilled water.
  • PCR reaction conditions 94 C predenaturation for 5 min; 94 °C denaturation for 30 s, 56 ⁇ annealing for 30 s, 72 °C for 30 s, 33 cycles; 72 °C for 10 min, the product was ligated by EcoRK Bglll digestion pCAMBIA2300 (promega T4 ligase cassette) was obtained as pCAMBIA2300-1.
  • Tnos was amplified using SEQ ID NO: 13 and SEQ ID NO: 14 with PBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, 33 cycles; extension at 72 °C for 10 min.
  • the product was cleaved by Sacl, EcoRI and ligated into pCAMBIA2300-1 to obtain pCAMBIA2300-2.
  • the Arabidopsis thaliana rd29A promoter was amplified using SEQ ID NO: 15 and SEQ ID NO: 16 using Arabidopsis thaliana (Columbia type, available from www.arabidopsis.org) genomic DNA as a template (see Zeng J., et L 2002, Preparation of total DNA from "recalcit rant plant taxa", Acta Bot. Sin., 44(6): Method 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase from TaKaRa was used.
  • PC reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ Arabidopsis DNA, 1.0 ⁇ PrimeSTAR 10 ⁇ primers SEQ ID NO: 15 and SEQ ID NO: 16 each 2.0 ⁇ l, and 31 ⁇ Double steamed water.
  • PCR reaction conditions predenaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, 33 cycles; extension at 72 °C for 10 min, PCR products through HindIII, Sail
  • the restriction enzyme was ligated to pCAMBIA2300-2 to obtain pCAMBIA2300-3.
  • the GhCBF2 gene (template was the pGEM T-easy recombinant vector containing the GhCBF2 gene obtained in Example 1) was amplified using SEQ ID NO: 17 and SEQ ID NO: 18, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ PCR reaction system: 10 ⁇ 5 ⁇ PS Buffer , 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ GhCBF2-pGEM, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 17 and SEQ ID NO: 18 each 2.0 ⁇ 1, and 31 ⁇ ⁇ double distilled water.
  • Agrobacterium tumefaciens LBA4404 (purchased from Biovector Science Lab, Inc) Competent preparation: Agrobacterium LBA4404 was sequenced on LB solid medium containing 5C ⁇ g/ml rifampicin and 5C ⁇ g/ml streptomycin 1 ⁇ 1d in advance Spot vaccination, cultured at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin and 50 ⁇ ⁇ / ⁇ 1 streptomycin, and cultured overnight (about 12-16 h) at 28 Torr until the OD600 value was 0.4, forming seed bacteria. liquid.
  • Transformation of Agrobacterium The competent cells were thawed on ice, and ⁇ rd29A-GhCBF2-2300 plasmid was added to 40 ⁇ l of competent cells, and the mixture was mixed and ice bathed for about 10 min. Transfer the mixture of competent and DNA to a pre-cooled electric shock cup with a gun and tap 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. When using a 0.1cm electric shock cup, the program of the MicroPulser (purchased from bio-rad) is set to "Agr" and the electric 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 gun. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h. Take 100 ⁇ 200 ⁇ 1 of bacterial solution coated with the corresponding resistance screening medium (LB solid medium, containing 50 ⁇ ⁇ / ⁇ 1 rifampicin, 50 ⁇ ⁇ / ⁇ 1 streptomycin, 50 ⁇ ⁇ / ⁇ 1 kanamycin) plate On, culture at 28 ° C.
  • LB solid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin, 50 ⁇ ⁇ / ⁇ 1 streptomycin, 50 ⁇ ⁇ / ⁇ 1 kanamycin
  • the sterile tobacco seeds were placed in MS solid medium (18.78 mM KN0 3 , 1.25 mM KH 2 P0 4 , 20.6 mM NH4NO3, 1.5 mM MgS0 4 , 3.0 mM CaCl 2 , 50 ⁇ M KI, 100 ⁇ M H3BO3 , 100 ⁇ M MnS0 4 , 30 ⁇ M ZnS0 4 , 1 M Na 2 Mo0 4 , 0.1 u M CoCl 2 , 100 ⁇ M Na 2 EDTA, 100 u M FeSO 4 , 7.4 g/L agar, sucrose 30 g L) Prepare sterile seedlings by sterile germination.
  • the leaves of the sterile seedlings were cut into 5 mm X 5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing the expression vector in the logarithmic growth phase for 10 min, and the bacterial liquid was absorbed and co-cultured for 2 days in the dark (MS medium). ).
  • the leaves were transferred to a differentiation medium (MS + 1 mg / L BA + 0.1 mg / L NAA + 50 mg / L kanamycin + 500 mg / L cephalosporin), cultured under light conditions for about 45 days, to grow buds
  • transfer to rooting medium MS+50mg/L kanamycin+500mg/L cephalosporin
  • the seedlings are transferred to only 500mg/L cephalosporin. The number was saved on the MS medium.
  • the obtained transgenic tobacco leaves were taken, and genomic DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and PCR was carried out using primers SEQ ID NO: 17 and SEQ ID NO: 18 (50 ⁇ PC reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 17 and SEQ ID NO: 18 each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • T Q generation transgenic tobacco T. L1, T Q L2, T. L3, T 0 L4 and T. L5 seeds and wild-type non-transgenic control tobacco seeds were separately sown on vermiculite, 25 ° C, 10 hours light culture / 14 hours dark culture cycle, 1/2 MS medium was poured every 5 days, after 25 days of culture, extract At the bottom of a leaf, genomic DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and primers SEQ ID NO: 17 and SEQ ID NO: 18 were used for PCR identification (reaction and conditions are the same as above), and negative plants were excluded (control) Tobacco also picks up a leaf).
  • transgenic tobacco (T ⁇ l, TjL2, T!L3, and each 10 strains, numbered 1" Mountain 1-1 to Dingshan 1-10, T!L2-1 to! ⁇ Mountain 2 10, T ⁇ -l to 1 ⁇ 13-10, T!L4-1 to 1 ⁇ 1 ⁇ 4-10 and ⁇ 5-1 to 1 ⁇ 1 ⁇ 5-10), control tobacco (10 strains) to make cold Experiment.
  • the sterilized vermiculite was soaked in 1/2 MS medium. Will be transgenic tobacco T. L1, T Q L2, T Q L3, T. The seeds of L4 and T Q L5 and the control tobacco seeds were separately sown on vermiculite, 25 ⁇ , 10 hours light culture/14 hours dark culture cycle, 1/2 MS was poured every 5 days, and after 25 days of culture, the bottom piece was taken.
  • TiL3 Dingshan 4 and Dingshan 5 each 10 strains, numbered from Dingshan 1-11 to 1 ⁇ Mountain 1-20, Dingshan 2-11 to 1 ⁇ Mountain 2-20, TiL3-ll to Dingshan 3-20 1"Mountain 4-11 to 1" Mountain 4-20 and 1"Mountain 5-11 to 1 ⁇ Mountain 5-20), and Control Tobacco (10 strains) for drought tolerance experiments.
  • the drought resistance identification of the above transgenic plants showed that the control plants were wilting, and the transgenic plants were able to grow normally, showing Significant drought resistance (Figure 4 and Table 1).
  • Figure 4 the selection of transgenic plants L5-11 and a control tobacco showed that the results of other tobaccos were similar.

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Abstract

提供了一个来源于棉花的DREB1类转录因子GhCBF2及其编码基因，以及其在培育耐逆性提高的转基因植物中的应用。

Description

棉花的一个 DREB1类转录因子及其编码基因与应用 技术领域 本发明涉及植物转录因子及其编码基因与应用， 特别是涉及一个来源于棉花的
DREB1类转录因子 GhCBF2及其编码基因， 以及其在培育耐逆性提高的转基因植物中的 应用。 背景技术 非生物胁迫， 如干旱、盐渍、极端温度、化学污染和氧损伤等能够对植物的生长发育 造成严重的危害， 对作物产量造成极大损失， 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 干早、 半干早地区占陆地面积的 34%; 我国干早、 半干旱地区约占国土面积的 52%, 年受 早面积达 20〜270万公顷 ， 全国灌溉区每年缺水约 30亿立方米， 因缺水而少收粮食 350〜 40亿公斤； 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。
由于植物的耐胁迫性大多属于数量性状， 现有可利用的种质资源匮乏， 采用常规育 种技术改良植物胁迫耐性的难度相当大， 培育出真正的耐胁迫品种就尤为困难。 近年来, 随着对植物抗逆分子机理研究的不断深入和分子生物学技术的迅猛发展， 抗逆研究已经 从生理水平深入到分子水平， 促进了植物抗逆基因工程的发展。当植物在受到胁迫时会产 生相应的应答反应， 来降低或消除给植株带来的危害。植物的这种应答反应是一个涉及多 基因、 多信号途径、 多基因产物的复杂过程。 这些基因及其表达产物可以分为 3 类： （1) 参与信号级联放大系统和转录控制的基因及产物； (2)直接对保护生物膜和蛋白质起作用 的基因及其表达产物； （3 ) 与水和离子的摄入和转运相关的蛋白质。 近年来， 通过转基 因技术提高植物对胁迫耐受能力的研究， 以及对胁迫具有耐受能力的农作物、旱生植物和 盐生植物的研究都取得了显著的成果， 对胁迫相关基因和信号转导系统也有了更进一歩 的了解 （Liu Q.1998. Two transcrip tion facto rs,DREBl and DREB2,w ith an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought-and low temperature-responsive gene exp ression, respectively, in A rabidopsis. Plant Cell, 10: 1391-1406； KAN GJY.2002. A rabid op sis basic leucine zipper p ro teins that mediate stress2responsive abscisic acid signaling。 Plant Cell, 14: 343- 357； ABE H.2003.A rabid op sis AtMYC2 (bHLH) and AtMYB2(MYB) function as transcrip tional activato rs in abscisic acid signaling. Plant Cell, 15: 63-78. ) 。
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占多 数， 但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机理还有待进 一步研究。 虽然许多研究机构通过现代生物技术， 获得了各类具有一定抗逆能力的转基 因植物， 但还未达到产业化的标准。 因此在提高植物抗逆性方面， 还有许多工作需要做。
发明内容 本发明人利用 SSH与 RACE相结合的方法克隆出了棉花的一个 DREB (脱水应答元 件结合蛋白， dehydration responsive element binding protein) 类转录因子 （本文命名为 GhCBF2) 的编码基因的 DNA序列。 并发现将其导入转基因植株后， 可明显改善转基因 植株的抗寒性和抗早性， 而且这些性状可稳定遗传。
本发明第一方面提供棉花的一个 DREB类转录因子 GhCBF2，其序列为 SEQ ID NO: l o
本发明第二方面提供编码本发明第一方面所述的转录因子的核苷酸序列。 优选地， 所述核苷酸序列具有 SEQ ID NO: 2所示的核苷酸序列。
本发明第三方面提供一种重组表达载体， 其含有本发明第二方面所述的核苷酸序列 并且所述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 rd29A-GhCBF2-2300载体。
本发明第四方面提供一种重组细胞， 其含有本发明第二方面所述的核苷酸序列或者 本发明第三方面所述的重组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。
本发明第五方面一种改善植物耐寒性和 /或抗旱性的方法， 包括： 将本发明第二方面 所述的核苷酸序列或者本发明第三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， 所述植物是烟草。
本发明第六方面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 含有本发明第二方面所述的核苷酸序列、 本发明第三方面所述的重组表达载体的植物或 植物组织； 优选地， 所述植物是烟草。
本发明第七方面提供本发明第一方面所述的转录因子、 本发明第二方面所述的核苷 酸序列、 本发明第三方面所述的重组表达载体或者本发明第四方面所述的重组细胞用于 改善植物耐寒性和 /或抗早性以及用于植物育种的用途； 优选地， 所述植物是烟草。 附图说明 图 1是 GhCBF2的植物表达载体rd29A-GhCBF2-2300)的构建流程。
图 2是 GhCBF2的植物表达载体 (rd29A-GhCBF2-2300)的质粒图。
图 3是转基因烟草抗寒性的实验结果。 图左为转基因植株（ L3-2); 图右为非转基因 植株 （对照）。
图 4是转基因烟草抗旱性的实验结果。 图左为转基因植株 （Τ^5-11); 图右为非转基 因植株 （对照）。 具体实施方式 实施例 下面结合非限制性实施例对本发明进行进一步说明。
下面实施例中提到的限制性内切酶均购自 NewEnglandBiolabs公司 实施例 1 冷胁迫下棉花 SSH文库构建：
具体方法为：
利用 Clontech公司的 PCR-selectTM cDNA Subtraction Kit, 按照产品说明书所示的方 法通过抑制消减杂交方法构建消减文库。在实验过程中以低温处理 6h的棉花幼苗叶片的 mRNA作为样本 (tester), 以未处理的棉花幼苗叶片的 mRNA作为对照 (driver)。
(1) 供试材料：
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 25Ό、光周期 16h/8h条件下培养，每周浇 1/2MS培养基（9.39 mM KN03， 0.625 mM KH2P04, 10.3 mM NH4N03, 0.75 mM MgS04, 1.5mMCaCl2， 50 μ M KI， 100μΜΗ3ΒΟ3， 100 MMnSO4, 30pMZnSO4， 1 MNa2Mo04, 0.1 M CoCl2, 100 MNa2EDTA, 100 MFeSO4) 一次。 当苗株高达 25-30cm时用于实验。
(2) 材料处理： 将供试幼苗分为 2组， 每组 4盆， 每盆 1株。 第一组为对照组， 在 25 °C、光照培养， 第二组为低温处理组， 在 4°C低温中处理 6h， 光照培养。 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 -70Ό冰箱中保存。
(3 ) 总 RNA提取：
分别取对照和低温处理的棉花叶子 0.5g，用植物 RNA提取试剂盒（invitrogen)提取 棉花的总 RNA。 用 HITACHI公司的紫外分光光度计 U-2001测定总 RNA在 260nm和 280nm的吸光度值， OD260/OD280比值为 1.8-2.0， 表明总 RNA纯度较高， 用 1.0%的琼 脂糖凝胶电泳检测总 RNA的完整性， 28S条带的亮度约为 18S条带的 2倍， 表明 RNA 的完整性良好。 使用 Qiagen公司的 Oligotex mRNA纯化试剂盒 (purification of polyA+ RNA from total RNA)分离 mRNA。
(4) 抑制消减杂交：
为了增加获得 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， 本实验室使用 Clontech公司的 PCR-selectTM cDNA Subtraction Kit, 按照商品说明书 用 Rsal， Haelll分别对来自上述分离 mRNA的双链 cDNA进行消化， 做两组抑制消减杂 交， 其他步骤及方法严格按 Clontech公司的 PCR-select™ cDNA Subtraction Kit产品说明 书所示的方法进行抑制消减杂交， 最后合并两组正向消减杂交 cDNA片段的第二次 PCR 产物。
( 5 ) cDNA消减文库的构建与初步筛选、 克隆、 鉴定
合并的正向消减杂交 cDNA片段的第二次 PCR产物 （QIAquick PCR Purification Kit 纯化， 购自 Qiagen) 与 pGEM-T Easy载体 (购自 Promega)连接， 依照 pGEM-T Easy试剂 盒产品说明书所示的方法，具体步骤如下：用 200ul PC 管依次加入下列成分：纯化 cDNA 片段的第二次 PCR产物 3ul， T4连接酶缓冲液 5 ul， pGEM-T Easy载体 l ul， T4 DNA连 接酶 lul， 于 4Ό连接过夜。 取 ΙΟμΙ^连接反应产物， 加入到 ΙΟΟμί 感受态大肠杆菌 JM109(购自 TAKARA)中， 冰浴 30min、 热休克 60s、 冰浴 2min， 另加 250 μL LB培养液 ( 1% Tryptone购自 OXOID, 0.5% Yeast Extract购自 OXOID, 1% NaCl购自国药）置 37 °C摇床中， 225 r/min摇菌 30min，取 200 μL菌液种植于含 50 ug/mL氨苄青霉素、 40 ug/mL X-gal、 24 ug/mL IPTG (X-gal/IPTG购自 TAKARA) 的 LB (同上） 固体培养平板上， 37 °C培育 18 ho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 200个白色 菌落 (编号： Gh-C001至 Gh-C200)于含有 50 ug/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 (PCR-select™ cDNA Subtraction Kit试剂盒自带） 禾 D Primer 2R (PCR-selectTM cDNA Subtraction Kit试剂盒自带）进行菌液 PCR扩增， 得到 152个阳性 克隆， 对所有阳性克隆在送英潍捷基 （上海） 贸易有限公司测序。
( 6 ) 差异克隆的 cDNA测序分析：
将 DNA测序结果去除载体和不明确序列及多余的 cDNA后， 共得到 112 个 EST(unigene；)。 经 BlastN发现其中 52条 unigene在 GenBank 中有同源序列 （相似 50% 以上）， 25条 EST功能未知或者为假定蛋白， 另有 35条未获得同源匹配， 推测可能是处 于 3 '、 5 ' 末端非翻译区的较短序列。 实施例 2 GhCBF2编码基因的克隆
在实施例 1有同源序列的 unigene中， 克隆子 Gh-C072序列如 SEQ ID NO: 3所示：
1 ACTTTTCTCA CAGCGGAGAT GGCAGCTCGT GCCCACGACG TAGCGGCCAT AGCTCTGAGG
61 GGGAGGTCGG CTTGCCTGAA CTTCGCGGAC TCAGCTTGGA GGCTTCCGGT TCCGGCTTCT
121 GCTGATCCCA AGGATATCCA GAAGGCGGCG GCAGAGGCGG CGGAGGCATT TAGACAACCG 181 GCTGAGCAAT GGGACGGAAA CTCTGAAGCT AATGCAAAGG GAGATGAAAA CAAAGGGAGT
241 TTGTGTGAGA ATGGGTTTTA TTTGGACGAG GAGGCGGTTT TTGGAACACA AAGTTATCTG
301 GAAAACATGG CGGCAGGGAT GATGATGTCA CCCCCTCGTT GTGGGT 序列分析表明该序列的编码的氨基酸序列属于 DREB1类蛋白，本文将该蛋白命名为 GhCBF2。
( 1 ) GhCBF2的全长基因的克隆
根据己经获得的 GhCBF2的基因片段， 设计两条特异性引物， 作为 3 ' RACE的 5 ' 端特异性引物：
G CBF2 GSP1 : SEQ ID NO: 4：
CTCACAGCGGAGATGGCAGCTC
GhCBF2 GSP2: SEQ ID NO: 5：
TCGTGCCCACGACGTAGCGGCCAT
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）
用 SEQ ID NO: 4与 3 '端引物 AUAP (试剂盒自带）， 以 mRNA逆转录的 cDNA为模 板进行第一轮 PCR扩增。 具体步骤如下： Ex Taq购自 TAKARA, 50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ mRNA反转录的 cDNA, 1.0 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 4和 AUAP各 2.0μ1，以及 35 μΐ的双蒸水。 PCR反应条件： 94V 预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 lmin， 33个循环； 72°C 延 伸 10 min。
所得的 PCR产物用双馏水稀释 50倍后取 1 μΐ作为模板，用 SEQ ID NO: 5与 3 '端引 物 AUAP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 2.5mM的 dNTP， 2.0 μΐ第一轮稀释的 PCR产物， 1.0 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 5和 AUAP各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min; 94V 变性 30 s， 58°C退火 30 s， 72 °C 延伸 lmin， 33个循环； 72°C 延伸 10 min。第二次 PCR 产物（QIAquick PCR Purification Kit纯化，购自 Qiagen) 3ul连接于 pGEM-T Easy Vector, 转化到大肠杆菌 JM109(具体方法同上)，随机挑取 10个白色菌落于含有 50 ug/mL氨苄青 霉素的 LB 液体培养基中培养， 37Ό培养过夜后加甘油至终浓度 20%, -80Ό保存备用。 SEQ ID NO: 5与 3 '端引物 AUAP进行菌液 PCR扩增 （体系及条件同上）， 得到 4个阳 性克隆， 送英潍捷基 （上海） 贸易有限公司测序,获得该基因的 cDNA的 3 '端。
根据已经获得的 GhCBF2的基因片段， 设计三条特异性引物， 分别作为 mRNA的反 转录引物 （SEQ ID NO: 6) 和 5 ' RACE的 3 ' 端特异性引物 （SEQ ID NO: 7和 8)。
G CBF2 GSP3 : SEQ ID O: 6：
AAACCGCCTC CTCGTCCAAA T G CBF2 GSP4: SEQ ID O: 7：
CCTCCCCCTC AGAGCTATGG C
GhCBF2 GSP5 : SEQ ID O: 8：
CGCTACGTCG TGGGCACGAG CT
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）。 SEQ ID NO: 6作为 mRNA反转录成 cDNA的引物。
用 SEQ ID NO: 7与 5'通用引物 AAP (试剂盒自带）， 以 mRNA逆转录的 cDNA (反 转录引物 SEQ ID NO: 6) 为模板进行第一轮 PCR扩增， 具体步骤如下： Ex Taq购自 TAKARA, 50 μΐ PCR反应体系： 5 μΐ lOxEx Buffer, 3 μΐ 2.5mM的 dNTP, 2.0 μΐ总 RNA 反转录的 cDNA， 1.0 μΙ Εχ Τας^ 10 μΜ的引物 SEQ ID NO: 7和 AAP各 2.0μ1， 以及 35 μΐ 的双蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 55 °C退火 30 s， 72 °C 延 伸 lmin， 33个循环； 72 "C 延伸 10 min。
所得的 PCR产物用双蒸水稀释 50倍后取 1 μΐ作为模板，用 SEQ ID NO: 8与 3 '端引 物 AUAP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 2.5mM的 dNTP， 2.0 μΐ第一轮稀释的 PCR产物， 1.0 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 8和 AUAP各 2.0μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 lmin， 33个循环； 72°C 延伸 10 min。第二次 PCR 产物（QIAquick PCR Purification Kit纯化，购自 Qiagen) 3ul连接于 pGEM-T Easy Vector, 转化到 JM109(具体方法同上)，随机挑取 10个白色菌落于含有 50 ug/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: 8 与 3'端引物 AUAP进行菌液 PCR扩增（反应体系及反应条件同上）， 得到 4个阳性克隆, 送广州英潍捷基 （上海） 贸易有限公司测序， 获得该基因的 cDNA的 5 '端。
所得的 5 ' RACE产物克隆测序后， 与 3 ' RACE产物测序结果拼接。 获得 GhCBF2 的全长 cDNA序列。 根据 GhCBF2的全长 cDNA序列设计一对引物如下：
G CBF2F: SEQ ID NO: 9：
ATGCTCGATTCTGGGTCTGTTTCGTCGCC
GhCBF2R: SEQ ID NO: 10：
TTATTGACAG TAACTCCAAA GAGGTAT
通过 SEQ ID NO: 9禾卩 SEQ ID NO: 10来克隆 GhCBF2编码基因的全长。
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶，以棉花的 cDNA为模板进行 PCR反应。
50 l PCR反应体系： 10 μ 1 5 X PS Buffer, 3 μ 1 2.5mM的 dNTP, 2.0 1 cDNA, Ι .Ο μ 1 PrimeSTAR, 10 μ M的引物 SEQ ID NO: 9禾卩 SEQ ID NO: 10各 2.0 μ 1， 30 μ ΐ的双蒸水。 PCR反应条件： 94°C预变性 5min; 94 °C 变性 30s， 58°C退火 30s， 72 °C 延伸 60s， 33 个循环； 72 °C 延伸 lOmino
PCR扩增产物加 A: 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 μ 1双蒸水溶解。加入 2.5ul 10 X Ex Buffer, 0.5ul 5 mM的 dATP ， 2.5ul lO X Ex Taq。 反应条件： 70°C反应 30分钟。 将得到约 600bp的 DNA片段回收 （Omega 回收试剂盒）， 克隆至 pGEM T-easy载体上， 转化 JM109(方法同上)。 随机挑取 10个白 色菌落于含有 50 ug/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: 9与 SEQ ID NO: 10进行菌液 PCR扩增 （反 应体系及反应条件同上）， 得到 5 个阳性克隆， 送英潍捷基 （上海） 贸易有限公司测序, 序列为 SEQ ID NO: 2。 其蛋白表达序列为 SEQ ID NO: 1。
GhCBF2的氨基酸序列： SEQ ID NO: 1
1 MLDSGSVSSP LSDSGSGTSR
21 PANFSDEDVM LAS ICPKKRA 4 1 GRKKFRETRH PVYRGVRRK
6 1 SGKWVCEVRE PNKKSRHG
81 TFLTAEMAAR AHDVAAIALJR
10 1 GRSACLNFAD SAWRLPVPAS
12 1 ADPKDIGKAA AEAAEAFRQP
14 1 AEQWDGNSEA NAKGDENKGS
16 1 LCENGFYLDE EAVFGTQSYL
18 1 ENMAAGMMMS PPRCGYNGDE
2 0 1 LEYDAADDFI PLWSYCQ*
GhCBF2编码基因的核苷酸序列： SEQ ID NO- 2
1 ATGCTCGATT CTGGGTCTGT TTCGTCGCCG TTATCGGATT CTGGAAGTGG TACTTCTCGT
61 CCGGCAAATT TCTCCGATGA AGATGTGATG TTAGCTTCGA TTTGTCCGAA GAAACGAGCG
121 GGACGGAAGA AATTCCGCGA GACTCGGCAT CCGGTTTACC GTGGAGTCCG CCGGAAGAAC
181 TCGGGGAAGT GGGTTTGTGA AGTGAGGGAG CCTAATAAGA AGTCGAGGAT TTGGCTGGGT
241 ACTTTTCTCA CAGCGGAGAT GGCAGCTCGT GCCCACGACG TAGCGGCCAT AGCTCTGAGG
301 GGGAGGTCGG CTTGCCTGAA CTTCGCGGAC TCAGCTTGGA GGCTTCCGGT TCCGGCTTCT
361 GCTGATCCCA AGGATATCCA GAAGGCGGCG GCAGAGGCGG CGGAGGCATT TAGACAACCG
421 GCTGAGCAAT GGGACGGAAA CTCTGAAGCT AATGCAAAGG GAGATGAAAA CAAAGGGAGT
481 TTGTGTGAGA ATGGGTTTTA TTTGGACGAG GAGGCGGTTT TTGGAACACA AAGTTATCTG
541 GAAAACATGG CGGCAGGGAT GATGATGTCA CCCCCTCGTT GTGGGTACAA TGGGGATGAA
601 CTAGAATACG ATGCTGCCGA TGATTTCATA CCTCTTTGGA GTTACTGTCA ATAA 实施例 3 GhCBF2植物表达载体构建
植物表达载体 rd29A- GhCBF2-2300构建流程如附图 1所示。
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 ΡΤΠ基因含双增强子的 35 S启动子， 以降低 ΡΤΠ蛋白在植物中的表达。选择诱导型启动子 rd29A及 Tnos作为 GhCBF2基因的启动 子和终止子。 具体步骤如下：
使用 SEQ ID NO: 11和 SEQ ID NO: 12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 ^ 5 xPS Buffer, 3 μΐ 2.5mM的 dNTP， 1.0 μΐ PBI121 , 1.0 μΐ PrimeSTA 、 Ι Ο μΜ的引物 SEQ ID NO: 11和 SEQ ID NO: 12各 2.0μ1， 以及 3 1 μΐ的双蒸水。 PCR反应 条件： 94 C预变性 5 min; 94 °C 变性 30 s， 56 Ό退火 30 s， 72 °C 延伸 30 s， 33个循环； 72 °C 延伸 10 min， 产物通过 EcoRK Bglll酶切连接到 pCAMBIA2300 (promega T4连接 酶盒)获得 pCAMBIA2300-l。
SEQ ID NO: 11 ：
GCAC GAATTC ATACAAATGGACGAACGGAT SEQ ID NO: 12： ATCC AGATCT AGATCCGGTGCAGATTATTTG
使用 SEQ ID NO: 13和 SEQ ID NO ： 14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 2.5mM的 dNTP, 1.0 μ1 ΡΒΙ121 , 1.0 μΐ Prime STAR、 10 μΜ的引物 SEQ ID NO: 13和 SEQ ID NO: 14 各 2.0μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C 退火 30 s， 72 °C 延伸 30 s， 33个循环； 72°C 延伸 10 min。 产物通过 Sacl、 EcoRI酶切 连接到 pCAMBIA2300-l， 获得 pCAMBIA2300-2。
SEQ ID O: 13：
AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID O: 14：
TCAGAATTC CCAGTGAATT CCCGATCTAG TA
使用 SEQ ID NO : 15 和 SEQ ID NO : 16 以拟南芥 （哥伦比亚型 ， 购自 www.arabidopsis.org)基因组 DNA为模板扩增拟南芥 rd29A启动子 （参考 Zeng J., et L 2002, Preparation of total DNA from"recalcit rant plant taxa", Acta Bot. Sin., 44(6): 694-697 中的方法提取拟南芥 DNA)。 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PC 反 应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5mM的 dNTP, 1.0 μΐ拟南芥 DNA， 1.0 μΐ PrimeSTAR 10 μΜ的引物 SEQ ID NO: 15和 SEQ ID NO: 16各 2.0μ1， 以及 31 μΐ的双蒸水。 PCR反 应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 30 s， 33个循 环； 72°C 延伸 10 min, PCR产物通过 HindIII、 Sail酶切连接到 pCAMBIA2300-2， 获得 pCAMBIA2300-3。
SEQ ID O: 15：
ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID O: 16：
TGAGTCGACTCCAAAGATT TTTTTCTTTC CAATAG
使用 SEQ ID NO: 17和 SEQ ID NO: 18扩增 GhCBF2基因 （模板是实施例 1所获 得含有 GhCBF2基因的 pGEM T-easy重组载体）， 采用 TaKaRa的 PrimeSTAR HS DNA 聚合酶。 50 μΐ PCR 反应体系： 10 μΐ 5xPS Buffer , 3 μΐ 2.5mM 的 dNTP , 1.0 μΐ GhCBF2-pGEM， 1.0 μΐ PrimeSTAR, 10 μΜ的引物 SEQ ID NO: 17禾 Π SEQ ID NO: 18各 2.0μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58°C 退火 30 s， 72 °C 延伸 lmin， 33个循环； 72°C 延伸 10 min。 通过 Sall、 Sacl酶切连接到 pCAMBIA2300-3 , 获得植物表达载体， rd29A- GhCBF2-2300。 SEQ ID O: 17：
TGAGTCGAC ATGCTCGATTCTGGGTCTGTTTCGTCGCC SEQ ID O: 18：
AAGGAGCTC TTATTGACAG TAACTCCAAA GAGGTAT 实施例 4 rd29A- GhCBF2-2300表达载体转化农杆菌
农杆菌 LBA4404 (购自 Biovector Science Lab,Inc)感受态制备： 提前 l-2d将农杆菌 LBA4404在含 5C^g/ml利福平和 5C^g/ml链霉素的 LB固体培养基上划单斑接种， 28 °C培 养 1至 2d。 挑取单菌落接种于 5ml含 50μ§/ηι1利福平和 50μ§/ηι1链霉素的 LB液体培养 基中， 28Ό下摇动培养过夜 (；约 12-16h)至 OD600值为 0.4， 形成种子菌液。取 5ml活化后 的菌液 （1 :20的比例） 接种于 100ml同样浓度抗生素的 LB液体培养基中， 28°C摇动培 养 2-2.5h至 OD600=0.8。 冰浴菌液 10min， 每隔 3min摇匀一次， 令细菌均匀进入休眠状 态。 于 4°C下 4000g离心 10min， 弃上清液； 加入一定量预冷 10%甘油重悬浮菌体, 4 °C下 4000g离心 10min， 收集沉淀； 用 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 40μ1/管将其分装， 于 -70°C保存备用。
转化农杆菌： 在冰上融化感受态细胞， 往 40μ1 的感受态细胞中加入 Ιμΐ 的 rd29A- GhCBF2-2300质粒， 混匀后冰浴约 10 min。 将感受态和 DNA的混合物用枪转移到预冷 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 0.1cm的电击杯的时 候， MicroPulser (购自 bio-rad) 的程序设置为 "Agr"， 电击一次 。 立即取出电击杯， 加 入 28°C预热的 LB培养基。 快速而轻柔的用枪将细胞打匀。 将悬浮液转入 1.5ml的离心 管， 28°C， 225rpm培养 lh。 取 100〜200μ1的菌液涂布与相应的抗性筛选培养基 （LB 固体培养基， 含 50μ§/ηι1利福平、 50μ§/ηι1链霉素、 50μ§/ηι1卡那霉素） 平板上， 28°C培 养。 实施例 5 利用农杆菌介导的转化法获得转基因烟草
用 75%酒精浸泡烟草种子 （国家烟草中期库， 获取单位中国农科院烟草所， 库编号 I5A00660) 30s, 再用 0.1%升汞浸泡 8min， 进行表面消毒。将消过毒的烟草种子置于 MS 固体培养基 （18.78 mM KN03， 1.25 mM KH2P04, 20.6 mM NH4NO3, 1.5 mM MgS04， 3.0 mM CaCl2， 50 μ M KI, 100 μ M H3BO3 , 100 μ M MnS04, 30 μ M ZnS04, 1 M Na2Mo04， 0.1 u M CoCl2, 100 μ M Na2EDTA, 100 u M FeSO4, 7.4 g/L琼脂，蔗糖 30g L) 上无菌发芽， 制备无菌苗。取无菌苗叶片剪成 5mm X 5mm大小的叶盘， 用处于对数生长 期的含表达载体的农杆菌浸染叶盘 10min， 吸干菌液， 在黑暗条件下共培养 2天（MS培 养基）。将叶片转到分化培养基 (MS+lmg/L BA+0.1mg/L NAA+50mg/L卡那霉素 +500mg/L 头孢霉素） 上， 光照条件下培养 45 天左右， 待芽长大后切下转移到生根培养基 (MS+50mg/L卡那霉素 +500mg/L头孢霉素） 中培养 30天左右， 待根系发达后将小苗转 入仅加有 500mg/L头孢霉素的 MS培养基上进行编号保存。
取获得的转基因烟草叶片，提取基因组 DNA(同实施例 3中拟南芥 DNA提取方法）， 用引物 SEQ ID NO: 17和 SEQ ID NO: 18进行 PCR鉴定（50 μΐ PC 反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5mM的 dNTP， 2.0 μΐ DNA, 1.0 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 17 和 SEQ ID NO: 18各 2.0μ1，以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min; 94 °C 变性 30 s， 58 °C退火 30 s, 72 °C 延伸 lmin， 33个循环； 72 °C 延伸 10 min)， 保存阳性 植株， 分别编号为 TQL1至 T。L20。 选取 TQL1-TQL5的种子进行。 实施例 6过表达 GhCBF2的转基因烟草 代的抗寒模拟实验及功能鉴定
将灭过菌的蛭石用 1/2MS培养基浸透。 将 TQ代转基因烟草 T。L1、 TQL2、 T。L3、 T0L4 和 T。L5的种子以及野生型非转基因对照烟草种子分别播种在蛭石上， 25 °C、 10小时光培 养 /14小时暗培养循环，每 5天浇一次 1/2MS培养基，培养 25天之后，摘取最下端一片叶子， 提取基因组 DNA (同实施例 3中拟南芥 DNA提取方法），利用引物 SEQ ID NO: 17和 SEQ ID NO: 18做 PCR鉴定（反应及条件同上）， 剔除阴性植株（对照烟草也同样摘取一片叶子）。 挑取大小一致的转基因烟草（T^l、 TjL2, T!L3 , 和 各 10株，编号分别是 1"山1-1 至丁山1-10、 T!L2-1至！^山2-10、 T^ -l至 1^13-10、 T!L4-1至 1^1^4-10和 Τ^5-1至 1^1^5-10)、 对照烟草（10株）做耐寒实验。 转基因烟草、 对照烟草， 4°C胁迫 72小时。 再经过 14天恢 复培养。 然后取出植株对植株进行称重。 上述 Ί代转基因植株的抗寒性鉴定表明， 对照 植株不能恢复正常生长， 生长明显受到抑制， 而转基因植株生长明显高于对照植株， 显 现出明显的抗寒性 （附图 3及表 1 )。 在图 3中， 选取转基因植株 和一株对照烟草示 例性显示， 其他烟草的结果与它们类似。 实施例 7过表达 GhCBF2的转基因烟草 ^代的抗旱模拟实验及功能鉴定
灭过菌的蛭石用 1/2MS培养基浸透。 将 代转基因烟草 T。L1、 TQL2、 TQL3、 T。L4和 TQL5的种子以及对照烟草种子分别播种在蛭石上， 25 Ό、 10小时光培养 /14小时暗培养循 环， 每 5天浇一次 1/2MS, 培养 25天之后， 摘取最下端一片叶子， 提取基因组 DNA (同实 施例 3中拟南芥 DNA提取方法）， 利用引物 SEQ ID NO: 17和 SEQ ID NO: 18做 PCR鉴定， 剔除阴性植株 (对照烟草也同样摘取一片叶子）。
Figure imgf000014_0001
TiL3 丁山4和丁山5各 10株， 编号分别是丁山1-11至1^山1-20、 丁山2-11至 1^山2-20、 TiL3-ll 至丁山3-20、 1"山4-11至 1"山4-20和 1"山5-11至 1^山5-20)、 对照烟草（10株）做耐旱实验。 转 基因烟草、 对照烟草干旱 14天 (不浇水)， 25 、 10小时光培养 /14小时暗培养循环。 然后 取出植株对植株进行称重。 上述 代转基因植株的抗旱性鉴定表明， 对照植株萎蔫严重， 而转基因植株能够正常生长， 显示出明显的抗旱性 （附图 4及表 1 )。 图 4中， 选取转基因 植株 L5-11和一株对照烟草示例性显示， 其他烟草的结果与它们类似。 表 1
Figure imgf000014_0002

Claims

权 利 要 求 书
1. 棉花的一个 DREB类转录因子， 其序列为 SEQ ID N0: 1。
2. 编码权利要求 1所述的转录因子的核苷酸序列。
3. 权利要求 2所述的核苷酸序列，其特征在于具有 SEQ ID NO: 2所示的核苷酸序列。
4. 一种重组表达载体， 其含有权利要求 2或 3所述的核苷酸序列并且所述核苷酸序 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 rd29A-GhCBF2-2300载体。
5. —种重组细胞， 其含有权利要求 2或 3所述的核苷酸序列或者权利要求 4所述的 重组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。
6. 一种改善植物耐寒性和 /或抗旱性的方法， 包括： 将权利要求 2或 3所述的核苷酸 序列或者权利要求 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， 所述植物是烟草。
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或 3所述的核苷酸序列或者权利要求 4所述的重组表达载体的植物或植物组织。
8. 权利要求 7所述的方法， 其中所述植物是烟草。
9. 权利要求 1的转录因子、 权利要求 2或 3所述的核苷酸序列、 权利要求 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寒性和 /或抗旱性以及用于 植物育种的用途。
10. 权利要求 9所述的用途， 其中所述植物是烟草。
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