WO2014190489A1 - Cotton dehydrin protein, coding gene of same, and application thereof - Google Patents

Cotton dehydrin protein, coding gene of same, and application thereof Download PDF

Info

Publication number
WO2014190489A1
WO2014190489A1 PCT/CN2013/076334 CN2013076334W WO2014190489A1 WO 2014190489 A1 WO2014190489 A1 WO 2014190489A1 CN 2013076334 W CN2013076334 W CN 2013076334W WO 2014190489 A1 WO2014190489 A1 WO 2014190489A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
seq
gene
expression vector
tobacco
Prior art date
Application number
PCT/CN2013/076334
Other languages
French (fr)
Chinese (zh)
Inventor
陈文华
孙超
何云蔚
崔洪志
Original Assignee
创世纪转基因技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 创世纪转基因技术有限公司 filed Critical 创世纪转基因技术有限公司
Priority to CN201380074552.0A priority Critical patent/CN105051059B/en
Priority to PCT/CN2013/076334 priority patent/WO2014190489A1/en
Publication of WO2014190489A1 publication Critical patent/WO2014190489A1/en

Links

Classifications

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

Definitions

  • the present invention relates to plant proteins and coding genes thereof and applications thereof, and in particular to a dehydrin protein derived from cotton dehydri n 09 and a coding gene thereof, and a resistance thereof in cultivation Application in transgenic plants with improved drought.
  • Dehydrin is a class of LEA proteins that are widely present in various tissues and organs of plants and in the late stages of plant embryo development. Dehydrin is a kind of highly hydrophilic protective protein synthesized by plants under the stress of abiotic stress such as low temperature, drought and high salt. It has the function of protecting nucleic acid, intracellular protein and membrane structure from damage. Many studies have confirmed that there is a close relationship between the expression and accumulation of plant dehydrin and plant stress resistance under abiotic stress.
  • Abiotic stresses such as drought, salt, extreme temperature, chemical pollution and oxygen damage, can cause serious damage to plant growth and development, causing great losses to crop yields.
  • the impact of drought on crop yields is in many natural adversities. It ranks first, 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 arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid regions account for 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares. Cubic meters, due to lack of water, less than 350 to 40 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the areas with the most water shortage in China, and the spring drought frequently reaches 10 years.
  • 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 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.
  • High-vegetation studies on stress tolerance, as well as studies on stress-tolerant crops, xerophytes, and halophytes have yielded significant results, as well as stress-related genes and signal transduction systems. Understanding (Liu Q.1998. Two transcription factors, DREB 1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought and low temperature responsive gene expression, respectively, in Arabid
  • the first aspect of the present invention provides a gene encoding a dehydrin protein dehydrin09 of cotton (designated herein as Ghdehydrin09) having a nucleotide sequence of 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, wherein the nucleotide sequence of the gene
  • the expression control sequence of the recombinant expression vector is operably linked; preferably, the expression vector is pCAMBIA2300; preferably, the recombinant expression vector is the rd29A-Ghdehydrin 09-2300 vector shown in Figure 2.
  • a 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: the first party of the present invention
  • the gene described or the recombinant expression vector of the second aspect of the invention is introduced into a plant or plant tissue and the gene is expressed; preferably, the plant is tobacco.
  • 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 tobacco.
  • a sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought tolerance of a plant and for use in plant breeding Use;
  • the plant is tobacco.
  • the seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, which has the amino acid sequence as shown in SEQ ID NO: 1.
  • Fig. 1 is a construction flow of a plant expression vector (rd29A-Ghdehydrin 09-2300) of the Ghdehydrin09 gene (Fig. 1 a-lb).
  • Figure 2 is a plasmid map of the plant expression vector (rd29A-Ghdehydrin 09-2300) of the Ghdehydrin09 gene.
  • FIG 3 is a G / 3 ⁇ 43 ⁇ 4 / z 7 ⁇ T Q generation of transgenic tobacco plants of gene (FIG, T Q 6; right, T 0 9) as a control and non-transgenic tobacco plant (left, CK) drought Simulation experiment (seedlings after 15 days of transplanted bud transplanting, drought for 14 days (without watering), 25 'C, 10 hours light culture/14 hours dark culture cycle).
  • Figure 4 shows the use of reverse transcription PCR for T Q transgenic tobacco plants and non-transgenic control plants.
  • M is Marker
  • 1-3 is a non-transgenic control tobacco plant
  • 4-6 is a T Q generation plant of transgenic tobacco with insignificant drought tolerance effect
  • 7-14 is a T Q generation plant of transgenic tobacco with significant drought tolerance effect.
  • 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 cotton seedlings treated with the drought was used as a sample (Tester) during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a control. 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, cultured at 25 ° C, photoperiod 16 h light / 8 h dark, and normally watered with 1/2 MS liquid medium.
  • the second group was the drought treatment group, cultured at 25 °C, photoperiod 16h light/8h dark condition, stopped watering, treated for 10 days, and then timely cut out the leaves of the top 1/3 of the two groups of seedlings, and quickly frozen with liquid nitrogen. 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 was extracted with the plant RNA extraction kit (Invitrogen).
  • the absorbance of total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001.
  • the OD 260 / OD 280 ratio was 1.8-2.0, indicating that the total RNA was of high purity and was condensed with 1.0% agarose.
  • Gel electrophoresis detected the integrity of total RNA.
  • the 28S band was approximately twice as bright as the 18S band, indicating good RNA integrity. Isolation of mRNAo using Qiagen's purification of poly A+ RNA from total RNA
  • the method performs suppression subtractive hybridization.
  • Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and subtraction hybridization was performed 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.
  • the gene is not cleavable and the obtained sequence is in the untranslated region.
  • the tester cDNA and Driver cDNA were digested with the endonuclease Haelll as described above and subjected to subtractive hybridization and PCR amplification. Finally, the two sets of forward subtractive hybridization cDNA fragments were merged for the second time.
  • the second PCR amplification product of the combined forward subtractive hybridization cDNA fragment (QIAquick PCR Purification Kit, purchased from Qiagen) was used according to the method described in the product specification of pGEM-T Easy kit (purchased from Promega).
  • the pGEM-T Easy vector was ligated as follows: The following components were sequentially added to a 200 ⁇ PCR tube: Purified combined positive subtractive hybridization cDNA fragment for the second inhibitory PCR product 3 ⁇ 1, 2 ⁇ 4 DNA ligase buffer 5 ⁇ l of liquid, pGEM-T Easy vector 1 ⁇ l, T4 DNA ligase 1 ⁇ l, and ligated overnight at 4 °C.
  • ⁇ ⁇ ligation reaction product added to 100 ⁇ L 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 ⁇ L LB medium (1% Tryptone was purchased from OXOID, 0.5% Yeast Extract was purchased from OXOID, 1% NaCl was purchased from Sinopharm), placed in a 37 °C shaker, shaken at 225 r/min for 30 min, and 200 ⁇ L was taken.
  • competent E. coli JM109 purchased from TAKARA
  • LB medium 1% Tryptone was purchased from OXOID
  • 0.5% Yeast Extract was purchased from OXOID
  • 1% NaCl was purchased from Sinopharm
  • the solution was applied to 50 g/mL ampicillin (purchased from Amresco), 40 g/mL X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside), 24 g/mL IPTG (isopropyl- ⁇ -D-thiogalactopyranoside) (X-gal and IPTG were purchased from TAKARA) on LB (ibid.) solid culture plates, incubated 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 360 white colonies (number: Gh-D001 to Gh-D360).
  • the stop codon TGA is already present in the Ghdehydrin09 gene fragment (SEQ ID No: 3), so only 5 'RACE is required to clone its full-length coding gene.
  • Ghdehydrin09 GSP2 SEQ ID NO: 5:
  • Ghdehydrin09 GSP3 SEQ ID NO: 6:
  • the experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • the cDNA was reverse transcribed from cotton mRNA (reverse transcription primer SEQ ID NO: 4), and then the Poly C tail was added according to the procedure in the above 5' RACE kit instructions, and the first round of PCR was carried out using the tailed product as a template.
  • the primers used are SEQ ID NO: 5 and the universal primer AAP (provided with the kit). The 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: 5.0 and AAP of 2.0 ⁇ l each, and 35 ⁇ 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 55 °C for 30 s, extension at 72 °C for 2 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 with SEQ ID NO: 6 and the universal primer AUAP, and the specific steps were as follows: 50 ⁇ l ⁇ Reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 ⁇ l ⁇ 10 ⁇ 10 ⁇ primer SEQ ID NO: 6 and AUAP each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • a fragment of about 500 bp was recovered from the second round of PCR product and ligated into the pGEM-T Easy vector, which was then transformed into E. coli JM109 competent cells (specific method as above). The transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin for screening. .
  • a pair of primers were designed according to the sequence of SEQ ID NO: 17 as follows:
  • Ghdehydrin09 SEQ ID NO: 7:
  • Ghdehydrin09R SEQ ID NO: 8:
  • the full-length coding sequence of Ghdehydrin09 was cloned by SEQ ID NO: 7 and SEQ ID NO: 8.
  • the PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
  • 50 ⁇ PCR reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR ⁇ 10 ⁇ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 ⁇ l, and 30 ⁇ 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 2 min), extension at 72 °C for 10 min.
  • the PCR amplification product was added with A tail: The PCR product was added with 2.5 times of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, and the supernatant was removed, dried, and dissolved in 21 ⁇ l of double distilled water. Add 2.5 ⁇ ⁇ Buffer, 0.5 ⁇ 5 mM dATP, l ⁇ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. Approximately 600 bp of DNA fragment was recovered (Omega Recovery Kit) and ligated into the pGEM T-easy vector (obtained Ghdehydrin09-pGEM plasmid), which was then transformed into E.
  • SEQ ID NO: 7 and SEQ ID NO: 8 were used for PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the obtained sequence was SEQ. ID NO: 2, the amino acid sequence of the encoded dehydrin09 protein is shown in SEQ ID NO: 1.
  • the plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the Pnos promoter was used to replace the ⁇ gene with a double enhancer.
  • the 35S promoter reduces the expression of prion protein in plants.
  • the inducible promoter rd29A and Tnos terminator were selected as promoters and terminators of the Gfrdefrydrm09 gene, respectively.
  • the construction process is shown in Figure 1.
  • Pnos was amplified using the plant expression vector PBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 ⁇ PCR reaction system: 10 ⁇ 5 > ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ 1 ⁇ 121, 1.0 ⁇ Prime STAR, 10 ⁇ primer SEQ ID NO: 9 P SEQ ID NO: 10 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 56 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was digested with EcoRI and Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase cassette) to obtain pCAMBIA2300-1.
  • Primers SEQ ID NO: 11 and SEQ ID NO: 12 were used to amplify Tnos using PBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ PCR reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ PBI121, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 ⁇ l, and 31 ⁇ Double distilled water.
  • PCR 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 obtained PCR product was ligated into lj pCAMBIA2300-l (Promega, T4 ligase cassette) to obtain pCAMBIA2300-2.
  • the Arabidopsis rd29A promoter was amplified using Arabidopsis thaliana (Columbia type, available from www.arabidopsis.org) DNA using primers SEQ ID NO: 13 and SEQ ID NO: 14 (see Zeng J., et L. 2002). , Preparation of total DNA from "recalcit rant plant taxa", Acta Bot. Sin., 44(6): The method in 694-697 extracts Arabidopsis DNA). PrimeSTAR HS DNA polymerase using TaKaRa.
  • PCR reaction system 10 ⁇ 5 PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ Arabidopsis DNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 13 and SEQ ID NO: 14 each 2.0 ⁇ l, and 31 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated by HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3
  • 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 2 min), extension at 72 °C for 10 min.
  • the obtained PCR product was ligated (ligation method as above) pCAMBIA2300-3 to obtain plant expression vector rd29A-Ghdehydrin09-2300 (Fig. 2).
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Competent preparation: Agrobacterium tumefaciens LBA4404 was spotted on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, 28 °C Incubate for 1 to 2 days. Pick a single colony inoculated in 5 ml containing 50 g/ml rifampicin and 50 g/ml The LB liquid medium of streptomycin was shaken overnight (about 12-16 h) at 28 ° C until the OD 6 (K) value was 0.4, and a seed bacterial liquid was formed.
  • the glycerol was repeatedly washed 3-4 times; the bacterial pellet was resuspended by adding 10% glycerol pre-cooled in an appropriate amount of ice bath to prepare LBA4404 competent cells, which were dispensed at 40 ⁇ M/tube and stored at -70 °C until use.
  • Transformation of Agrobacterium LBA4404 competent cells were thawed on ice, and 1 ⁇ of the positive rd29A-Ghdehydrin09-2300 plasmid obtained in Example 3 was added to 40 ⁇ of the competent cells, and the mixture was mixed and ice bathed for about 10 min. The mixture of the competent cells after ice bath and the positive rd29A-Ghdehydrin 09-2300 plasmid was transferred to an ice-cold electric shock cup with a micropipette, and tapped to bring the suspension to the bottom, taking care not to have air bubbles.
  • the leaf discs of 5 mm ⁇ 5 mm size were inoculated with the Agrobacterium LBA4404 positive transformation clone containing the expression vector rd29A-Ghdehydrin 09-2300 obtained in Example 4 in the logarithmic growth phase for 10 min, and the bacterial solution was aspirated. , co-cultured for 2 days in dark conditions (MS medium).
  • the leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (NAA) + 50 mg/L kanamycin + 500 mg/L cephalosporin).
  • the cells were cultured for about 45 days under light conditions.
  • the obtained transgenic tobacco leaves were taken, DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and SEQ ID NO: 7 and SEQ ID NO: 8 (50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ l 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ ⁇ ⁇ , 10 ⁇ primer SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions 94 °C pre-denaturation 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min, and numbering of plants identified as positive by PCR (T Q 1 - T Q 20) and preserved.
  • T Q 1 - T Q 20 numbering of plants identified as positive by PCR
  • the sterilized vermiculite was soaked in 1/2 MS liquid medium.
  • the T Q 1-T Q 20 transgenic tobacco and the control tobacco (non-transgenic) tissue culture seedlings obtained in Example 5 were transplanted to vermiculite, respectively, at 25 ° C, 10 h light culture / 14 h dark culture cycle, every 5 days.
  • a 1/2 MS liquid medium was poured, and the drought stress test was carried out 15 days after the strong seedling culture.
  • the transgenic tobacco and the control tobacco were dried for 14 days (without watering), 25 ° C, 10 h light culture / 14 h dark culture cycle.
  • T Q transgenic plants showed that the control plants were severely wilted, while T Q 1 , T 0 3, ⁇ 0 5, ⁇ 0 6 , ⁇ 0 8 , ⁇ 0 9 , ⁇ 0 12, ⁇ .
  • Fifteen transgenic plants were able to grow normally and showed significant drought tolerance (see Figure 3, taking T Q 6, T Q 9 as an example, the rest not shown).
  • Example 7 The expression of the G/3 ⁇ 4 ⁇ fe/z 3 ⁇ 4& ⁇ 9 gene was verified at the transcriptional level.
  • RNA extracted by plant RNA extraction kit (Invitrogen) with 0.05 g of leaves per day for 14 days.
  • the absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the respective RNA concentrations.
  • Ll box Superscript III Reverse Transcriptase was used for reverse transcription (2 ⁇ g total RNA as template, reverse transcription primer SEQ ID NO: 8).
  • the relative expression of dehydrin09 protein was detected by amplifying Ghdehydrin09 by SEQ ID NO: 7 and SEQ ID NO: 8.
  • PCR was carried out using TaKaRa's PrimeSTAR HS DNA polymerase using the cDNA obtained by the above reverse transcription as a template.
  • 50 ⁇ PCR reaction system 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 ⁇ l, and 30 ⁇ Double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 29 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-3 is non-transgenic control tobacco, and 4-6 is transgenic tobacco T with insignificant drought tolerance.
  • Q- generation plants, 7-14 are transgenic tobacco T Q plants with significant drought tolerance (in order: ⁇ 0 1, ⁇ . 3, ⁇ . 5, ⁇ . 6, ⁇ . 8, T 0 9, T 0 12 , T 0 15 ).
  • the size of the band shown is consistent with the size of Ghdehydrin09 (approximately 600 bp).

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

A cotton-sourced dehydrin protein dehydrin09, a coding gene of same, and an application thereof in cultivating a transgenic plant of increased drought tolerance.

Description

一种棉花脱水素蛋白及其编码基因与应用 技术领域 本发明涉及植物蛋白及其编码基因与应用，特别是涉及一个来源于棉花的脱 水素蛋白 dehydrin09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。 技术背景 脱水素是 LEA蛋白中的一类，广泛存在于植物的各个组织器官及植物胚胎发 育后期。脱水素是植物在受低温、干旱和高盐等非生物逆境胁迫时合成的一类高 亲水性保护蛋白， 具有保护核酸、胞内蛋白和膜结构免受损害的功能。许多研究 已经证实在非生物胁迫下，植物脱水素的表达与积累和植物抗逆性之间存在着紧 密的联系。 FIELD OF THE INVENTION The present invention relates to plant proteins and coding genes thereof and applications thereof, and in particular to a dehydrin protein derived from cotton dehydri n 09 and a coding gene thereof, and a resistance thereof in cultivation Application in transgenic plants with improved drought. BACKGROUND OF THE INVENTION Dehydrin is a class of LEA proteins that are widely present in various tissues and organs of plants and in the late stages of plant embryo development. Dehydrin is a kind of highly hydrophilic protective protein synthesized by plants under the stress of abiotic stress such as low temperature, drought and high salt. It has the function of protecting nucleic acid, intracellular protein and membrane structure from damage. Many studies have confirmed that there is a close relationship between the expression and accumulation of plant dehydrin and plant stress resistance under abiotic stress.
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。  Abiotic stresses, such as drought, salt, extreme temperature, chemical pollution and oxygen damage, can cause serious damage to plant growth and development, causing great losses to crop yields. The impact of drought on crop yields is in many natural adversities. It ranks first, 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 arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid regions account for 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares. Cubic meters, due to lack of water, less than 350 to 40 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the areas with the most water shortage in China, and the spring drought frequently reaches 10 years.
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， 以及对具有胁迫耐受能力的农作物、 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 更进一步的了解 (Liu Q.1998. Two transcription factors,DREB 1 and DREB2,with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought and low temperature responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10: 1391-1406； KANGJY.2002. Arabidopsis basic leucine zipper proteins that mediate stress responsive abscisic acid signaling. Plant Cell, 14: 343- 357； ABEH.2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2(MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 15 : 63-78. ) 。 Since the stress tolerance of plants is mostly quantitative, the available germplasm resources are scarce. It is very difficult to improve the stress tolerance of plants by conventional breeding techniques. It is especially difficult to cultivate true stress-tolerant varieties. In recent years, with the deepening of research on the molecular mechanism of plant stress resistance and the rapid development of molecular biology technology, stress resistance research has progressed from physiological level to molecular level, which promoted the development of plant stress resistance genetic engineering. When plants are stressed, they will respond accordingly to reduce or eliminate the damage to plants. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. These 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. In recent years, through genetic modification technology High-vegetation studies on stress tolerance, as well as studies on stress-tolerant crops, xerophytes, and halophytes have yielded significant results, as well as stress-related genes and signal transduction systems. Understanding (Liu Q.1998. Two transcription factors, DREB 1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought and low temperature responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10: 1391-1406; KANGJY.2002. Arabidopsis basic leucine zipper proteins that mediate stress responsive abscisic acid signaling. Plant Cell, 14: 343-357; ABEH.2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2(MYB) function as transcriptional activators 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，许多植物对逆境的生物 化学和生理学上的响应机制仍有待深入研究。 在抗逆应答基因的功能及表达调 控方面的研究将为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脱水素蛋白 （本文命名为 dehydrin09 ) 的编码基 因， 并测定了其 DNA序列。 并发现将其导入受体植株并超量表达后， 可显著 改善受体植株的耐旱性， 而且这些性状可稳定遗传。  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. Research on the function and expression regulation of stress-responsive genes will provide an important basis for the link between stress-resistance-related signaling pathways and the study of the entire signaling network system. SUMMARY OF THE INVENTION The present inventors cloned a gene encoding a dehydrin protein of cotton (designated herein as dehydrin09) by SSH (suppression subtractive hybridization) and RACE (rapid amplification of cDNA ends), and determined the DNA sequence thereof. . It was also found that when introduced into recipient plants and overexpressed, the drought tolerance of the recipient plants was significantly improved, and these traits were stably inherited.
本发明第一方面提供棉花的一个脱水素蛋白 dehydrin09的编码基因 （本文 将其命名为 Ghdehydrin09 )， 其核苷酸序列为 SEQ ID NO: 2。  The first aspect of the present invention provides a gene encoding a dehydrin protein dehydrin09 of cotton (designated herein as Ghdehydrin09) having a nucleotide sequence of SEQ ID NO: 2.
本发明第二方面提供一种重组表达载体， 其含有本发明第一方面所述的基 因， 其是通过将所述基因插入到一种表达载体而获得的， 其中所述基因的核 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， 所述表 达载体是 pCAMBIA2300 ; 优选地， 所述重组表达载体为附图 2 所示的 rd29A-Ghdehydrin09-2300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, wherein the nucleotide sequence of the gene The expression control sequence of the recombinant expression vector is operably linked; preferably, the expression vector is pCAMBIA2300; preferably, the recombinant expression vector is the rd29A-Ghdehydrin 09-2300 vector shown in Figure 2.
本发明第三方面提供一种重组细胞， 其含有本发明第一方面所述的基因或 者本发明第二方面所述的重组表达载体； 优选地， 所述重组细胞为重组农杆菌 细胞。  A 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， 所述植物是烟草。 A fourth aspect of the present invention provides a method for improving drought tolerance of a plant, comprising: the first party of the present invention The gene described or the recombinant expression vector of the second aspect of the invention is introduced into a plant or plant tissue and the gene is expressed; preferably, the plant is tobacco.
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， 所述植物是烟草。  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 tobacco.
本发明第六方面提供本发明第一方面所述的基因、 本发明第二方面所述的 重组表达载体或者本发明第三方面所述的重组细胞用于改善植物耐旱性以及用 于植物育种的用途； 优选地， 所述植物是烟草。  A sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought tolerance of a plant and for use in plant breeding Use; Preferably, the plant is tobacco.
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 Ghdehydrin09基因的植物表达载体（rd29A-Ghdehydrin09-2300)的构 建流程 （图 l a-lb)。  The seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, which has the amino acid sequence as shown in SEQ ID NO: 1. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector (rd29A-Ghdehydrin 09-2300) of the Ghdehydrin09 gene (Fig. 1 a-lb).
图 2是 Ghdehydrin09基因的植物表达载体（rd29A-Ghdehydrin09-2300)的质 粒图。  Figure 2 is a plasmid map of the plant expression vector (rd29A-Ghdehydrin 09-2300) of the Ghdehydrin09 gene.
图 3是 G/¾¾/z 7^基因的 TQ代转基因烟草植株 （中图， TQ6; 右图， T09 ) 和作为对照的非转基因烟草植株（左图， CK) 的耐旱模拟实验（转化芽移栽 15 天后的幼苗， 干旱 14天 （不浇水）， 25 'C、 10小时光培养 /14小时暗培养循环） 结果。 FIG 3 is a G / ¾¾ / z 7 ^ T Q generation of transgenic tobacco plants of gene (FIG, T Q 6; right, T 0 9) as a control and non-transgenic tobacco plant (left, CK) drought Simulation experiment (seedlings after 15 days of transplanted bud transplanting, drought for 14 days (without watering), 25 'C, 10 hours light culture/14 hours dark culture cycle).
图 4是利用反转录 PCR对 TQ代转基因烟草植株和非转基因对照植株中Figure 4 shows the use of reverse transcription PCR for T Q transgenic tobacco plants and non-transgenic control plants.
G/¾¾/z ¾& ^9基因在转录水平上的分子检测的验证结果。 M为 Marker, 1-3为非 转基因对照烟草植株, 4-6为耐旱效果不显著的转基因烟草 TQ代植株, 7-14为耐旱 效果显著的转基因烟草 TQ代植株。 具体实施方式 下面结合非限制性实施例对本发明进行进一步说明。 Validation of molecular detection of the G/3⁄43⁄4/z 3⁄4& ^9 gene at the transcriptional level. M is Marker, 1-3 is a non-transgenic control tobacco plant, 4-6 is a T Q generation plant of transgenic tobacco with insignificant drought tolerance effect, and 7-14 is a T Q generation plant of transgenic tobacco with significant drought tolerance effect. 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 endonucleases mentioned in the examples below were purchased from New England Biolabs. Example 1 Construction of cotton SSH library 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), 以未处理的棉花幼苗的 叶片的 mRNA作为对照 （Driver)。 具体步骤如下：  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 cotton seedlings treated with the drought was used as a sample (Tester) during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a control. 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、 光照培养 （光周期 16h光照 /8h黑暗）， 每周 浇 1/2MS液体培养基（含 9.39 mM KN03， 0.625 mM KH2P04， 10.3 mM H4N03, 0.75 mM MgSO4， 1.5 mM CaCl2， 50 μΜ ΚΙ, 100 μΜ Η3ΒΟ3， 100 M MnSO4， 30 M ZnS04， 1 μΜ Να2Μο04, 0.1 M CoCl2， 100 μΜ Na2EDTA, 100 M FeS04， 余量为水） 一次。 当苗株高达 25-30cm时用于实验。 冀棉14 (National Cotton Medium-term Library, obtained by the China Cotton Research Institute, Uniform No.: ZM-30270) Seeded onto the sterilized vermiculite, cultured at 25 ° C, light (light cycle 16h light / 8h dark), each 1/2MS liquid medium (containing 9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM H 4 N0 3 , 0.75 mM MgSO 4 , 1.5 mM CaCl 2 , 50 μΜ ΚΙ, 100 μΜ Η 3 ΒΟ 3 ) 100 M MnSO 4 , 30 M ZnS0 4 , 1 μΜ Να 2 Μο0 4 , 0.1 M CoCl 2 , 100 μΜ Na 2 EDTA, 100 M FeS0 4 , balance water) 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， 用 1/2MS液体培养基正常浇灌。 第二组 为干旱处理组， 25°C、 光周期 16h光照 /8h黑暗条件下培养， 停止浇灌， 处理 10天， 然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, cultured at 25 ° C, photoperiod 16 h light / 8 h dark, and normally watered with 1/2 MS liquid medium. The second group was the drought treatment group, cultured at 25 °C, photoperiod 16h light/8h dark condition, stopped watering, treated for 10 days, and then timely cut out the leaves of the top 1/3 of the two groups of seedlings, and quickly frozen with liquid nitrogen. 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 (Invitrogen)提取棉花的总 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 18S条带的 2倍， 表明 RNA的完整性良好。 使用 Qiagen 公司 的 Oligotex mRNA纯化试剂盒 (purification of poly A+ RNA from total RNA)分离 mRNAo The cotton leaves of the control and drought-treated groups were each 0.5 g, and the total RNA of cotton was extracted with the plant RNA extraction kit (Invitrogen). The absorbance of total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001. The OD 260 / OD 280 ratio was 1.8-2.0, indicating that the total RNA was of high purity and was condensed with 1.0% agarose. Gel electrophoresis detected the integrity of total RNA. The 28S band was approximately twice as bright as the 18S band, indicating good RNA integrity. Isolation of mRNAo 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™ cDNA 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。 According to Clontech's PCR-selectTM cDNA Subtraction Kit kit instructions The method performs suppression subtractive hybridization. Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and subtraction hybridization was performed 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进行酶切并且 进行差减杂交和 PCR扩增。 最后合并两组正向差减杂交 cDNA片段的第二次 In order to increase the validity of the Expressed Sequence Tag (EST) (Unigene), the gene is not cleavable and the obtained sequence is in the untranslated region. In addition to the Rsal enzyme, the tester cDNA and Driver cDNA were digested with the endonuclease Haelll as described above and subjected to subtractive hybridization and PCR amplification. Finally, the two sets of forward subtractive hybridization cDNA fragments were merged for the second time.
PCR产物。 PCR product.
( 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扩增产物 （QIAquick PCR Purification Kit纯化， 购自 Qiagen)与 pGEM-T Easy载体连接， 具体步骤如下： 在 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 μ1， 于 4°C连接过夜。 然后， 取 ΙΟ μί连接反应产 物， 加入到 100 μL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 LB培养液 ( 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,取 200 μ 菌液涂布于含 50 g/mL氨苄青霉 素（购自 Amresco)、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 > l mm的清晰 白色及蓝色菌落数， 随机挑取 360个白色菌落 (编号： Gh-D001至 Gh-D360)。 将所有白色菌落挑于含有 50 μ§/ηΛ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%, 然后于 -80°C保 存备用。以巢式 PCR 弓 I物 Primer 1和 Primer 2R( Clontech公司的 PCR-selectTM cDNA Subtraction Kit试剂盒自带） 对所述培养所得的菌液进行 PCR扩增验证， 得到 238个阳性克隆，将所有阳性克隆送英潍捷基 （上海） 贸易有限公司测序 ( 6 ) 差异克隆的 cDNA测序分析： The second PCR amplification product of the combined forward subtractive hybridization cDNA fragment (QIAquick PCR Purification Kit, purchased from Qiagen) was used according to the method described in the product specification of pGEM-T Easy kit (purchased from Promega). The pGEM-T Easy vector was ligated as follows: The following components were sequentially added to a 200 μΐ PCR tube: Purified combined positive subtractive hybridization cDNA fragment for the second inhibitory PCR product 3 μ1, 2χΤ4 DNA ligase buffer 5 μl of liquid, pGEM-T Easy vector 1 μl, T4 DNA ligase 1 μl, and ligated overnight at 4 °C. Then, 反应 μί ligation reaction product, added to 100 μL 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 LB medium (1% Tryptone was purchased from OXOID, 0.5% Yeast Extract was purchased from OXOID, 1% NaCl was purchased from Sinopharm), placed in a 37 °C shaker, shaken at 225 r/min for 30 min, and 200 μL was taken. The solution was applied to 50 g/mL ampicillin (purchased from Amresco), 40 g/mL X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), 24 g/mL IPTG (isopropyl-β-D-thiogalactopyranoside) (X-gal and IPTG were purchased from TAKARA) on LB (ibid.) solid culture plates, incubated 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 360 white colonies (number: Gh-D001 to Gh-D360). All white colonies were picked in 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 μ § /ηΛ ampicillin, cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and then at -80 ° C Guarantee Save for backup. Nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) were used to perform PCR amplification on the cultured cells, and 238 positive clones were obtained, all positive. Cloning was sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing (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 134 条表达序列标签 （Expressed sequence tag, EST) (Unigene)。 经 BlastN发现其 中 79条 Unigene在 GenBank 中有同源序列（蛋白同源性 50%以上）， 30条 EST 功能未知或者为假定蛋白， 另有 25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 棉花脱水素蛋白基因 dehydrin09的克隆  After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 134 Expressed sequence tags (EST) (Unigene) were obtained. Among BlastN, 79 Unigenes have homologous sequences in GenBank (more than 50% protein homology), 30 EST functions are unknown or hypothetical proteins, and another 25 have not obtained homologous matches, presumably at 3 ' Or a shorter sequence of untranslated regions at the 5' end. Example 2 Cloning of cotton dehydrin protein gene dehydrin09
将编号为 Gh-D222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脱水素蛋白，本文将 SEQ ID No : 3 对应的全长编码基因命名为 Ghdehydrin09， 其对应的蛋白命名为 dehydrin09。  After the sequencing result of the positive clone numbered Gh-D222 was removed from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the protein encoded by the sequence belonged to the dehydrin protein, and the full length corresponding to SEQ ID No: 3 was herein. The coding gene was named Ghdehydrin09, and its corresponding protein was named dehydrin09.
SEQ ID No: 3 SEQ ID No: 3
1 CAGCTCTTCA AGCGACGAGG AAGAAGGTGA AGGAGAAGAG AAGAAGAAGA AGAAAAAGAA 1 CAGCTCTTCA AGCGACGAGG AAGAAGGTGA AGGAGAAGAG AAGAAGAAGA AGAAAAAGAA
61 GGAGAAGAAG GGACTGGAAG AAAAGATTGA AGAGAAATTA GAAGGGGAAA AGAAAGAAGA61 GGAGAAGAAG GGACTGGAAG AAAAGATTGA AGAGAAATTA GAAGGGGAAA AGAAAGAAGA
121 GGACACCTCA GTTCCAGTAG AGAAGTGCGA TGAGCCAGTA GTCCAGCCAG AGACGCCAGA 181 GAAGAAAGGT TTCCTCGAGA AGATCAAGGA GAAACTCCCT GGACAACACA AGAAAGCTGA 241 AGAGGCCAGT AGCCCGGCTC CAGCCCCAGC AGCTGAGCCC CATCACGAAG AGGAGTCAAA 301 GGAAAAGAAG GGAATTTTGG AGAAAATCAA GGAGAAGCTT CCTGGTTACC ACTCCAAATC 361 AGATGAAGAA AAAGAAAAGG CTTGAGATTC AAGTTCACAT AAAAATACTG ATGGGATTGT 421 GTGTTTCTTA AATTGTTCAA AAATTATCTG TTTTTGTTTT TGAGGTTGTA TTTGTTGTAT 481 ATTTCATATT TAAGTTTCTT CTTTTCCCAG CATTTTTAGT ACAAATCATA AGGAAAAAAA 541 AAAGTTTGCT TT 121 GGACACCTCA GTTCCAGTAG AGAAGTGCGA TGAGCCAGTA GTCCAGCCAG AGACGCCAGA 181 GAAGAAAGGT TTCCTCGAGA AGATCAAGGA GAAACTCCCT GGACAACACA AGAAAGCTGA 241 AGAGGCCAGT AGCCCGGCTC CAGCCCCAGC AGCTGAGCCC CATCACGAAG AGGAGTCAAA 301 GGAAAAGAAG GGAATTTTGG AGAAAATCAA GGAGAAGCTT CCTGGTTACC ACTCCAAATC 361 AGATGAAGAA AAAGAAAAGG CTTGAGATTC AAGTTCACAT AAAAATACTG ATGGGATTGT 421 GTGTTTCTTA AATTGTTCAA AAATTATCTG TTTTTGTTTT TGAGGTTGTA TTTGTTGTAT 481 ATTTCATATT TAAGTTTCTT CTTTTCCCAG CATTTTTAGT ACAAATCATA AGGAAAAAAA 541 AAAGTTTGCT TT
Ghdehydrin09全长编码基因的克隆 Cloning of the full-length coding gene of Ghdehydrin09
已经获得的 Ghdehydrin09基因片段（ SEQ ID No: 3 )中已经有终止密码子 TGA,因此只需要做 5 'RACE以克隆其全长编码基因。  The stop codon TGA is already present in the Ghdehydrin09 gene fragment (SEQ ID No: 3), so only 5 'RACE is required to clone its full-length coding gene.
根据已经获得的 SEQ ID No: 3序列， 设计三条特异性引物， 作为反转录 弓 I物及 5'RACE的 3 '端特异性引物。 Ghdehydrin09 GSP 1： SEQ ID NO: 4： Based on the sequence of SEQ ID No: 3 that has been obtained, three specific primers were designed as the reverse transcription primer and the 3'-end specific primer of 5' RACE. Ghdehydrin09 GSP 1: SEQ ID NO: 4:
TATGATTTGT ACTAAAAATG  TATGATTTGT ACTAAAAATG
Ghdehydrin09 GSP2： SEQ ID NO: 5：  Ghdehydrin09 GSP2: SEQ ID NO: 5:
TCTTGTGTTG TCCAGGGAGT TTC  TCTTGTGTTG TCCAGGGAGT TTC
Ghdehydrin09 GSP3： SEQ ID NO: 6：  Ghdehydrin09 GSP3: SEQ ID NO: 6:
TCTGGCGTCT CTGGCTGGAC  TCTGGCGTCT CTGGCTGGAC
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 experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
以棉花 mRNA反转录得到 cDNA (反转录引物 SEQ ID NO:4 ) , 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: 5与通用引物 AAP (试剂盒自带）， 具体步骤如下：  The cDNA was reverse transcribed from cotton mRNA (reverse transcription primer SEQ ID NO: 4), and then the Poly C tail was added according to the procedure in the above 5' RACE kit instructions, and the first round of PCR was carried out using the tailed product as a template. For amplification, the primers used are SEQ ID NO: 5 and the universal primer AAP (provided with the kit). The specific steps are as follows:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ mRNA 反转录并加 Poly C尾后的 cDNA、 1.0 μΐ Ex Taq (购自 TAKARA)、 10 μΜ的引 物 SEQ ID NO: 5和 AAP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， 55 °C退火 30 s， 72 °C 延伸 2min)， 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: 5.0 and AAP of 2.0 μl each, and 35 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 55 °C for 30 s, extension at 72 °C for 2 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: 6与通用引物 AUAP进行第二轮 PCR扩增， 具体步骤如下： 50 μ1 ΡΟ 反应体 系： 5 μΐ ΙΟ Εχ Buffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ稀释的第一轮 PCR产物、 1.0 μ1 Εχ Τα 10 μΜ的引物 SEQ ID NO: 6和 AUAP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 2 min) , 72 °C 延伸 10 min。 从第二轮 PCR产物中回收片 段约为 500 bp的条带 （Gel Extraction Kit购自 OMEGA) 连接于 pGEM-T Easy 载体， 然后将其转化到大肠杆菌 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 g/mL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。 SEQ ID NO: 6 与通用引物 AUAP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,送英潍捷基 （上海） 贸易有限公司测序,获得该基因的 cDNA的 5 ' 山 将所得的 5'RACE产物中的阳性克隆测序所得序列与 SEQ ID No: 3拼接， 获得 SEQ ID NO: 17： 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 with SEQ ID NO: 6 and the universal primer AUAP, and the specific steps were as follows: 50 μl ΡΟ Reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 μl Εχ 10α 10 μΜ primer SEQ ID NO: 6 and AUAP each 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 2 min), extension at 72 °C for 10 min. A fragment of about 500 bp (Gel Extraction Kit from OMEGA) was recovered from the second round of PCR product and ligated into the pGEM-T Easy vector, which was then transformed into E. coli JM109 competent cells (specific method as above). The transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin for screening. . 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. SEQ ID NO: 6 was subjected to bacterial liquid PCR amplification with the universal primer AUAP (reaction system and reaction conditions are the same as above), and 3 was obtained. A positive clone was sequenced and sent to the British singapore (Shanghai) Trading Co., Ltd. to obtain the cDNA of the gene. The sequence of the positive clone in the 5' RACE product obtained by sequencing the cDNA was sequenced with SEQ ID No: 3 to obtain SEQ. ID NO: 17:
1 GCGTTCCATT CCATTTCCGT ATAGTTCGAA AACACTTAAG TACCTTCGAT CATATTTTAG1 GCGTTCCATT CCATTTCCGT ATAGTTCGAA AACACTTAAG TACCTTCGAT CATATTTTAG
61 TGTCAAAAAA TGGCCGAGGA GCATACCAGC AAGGCCCCTG AGTTGGAGAG CAACGTTAGC61 TGTCAAAAAA TGGCCGAGGA GCATACCAGC AAGGCCCCTG AGTTGGAGAG CAACGTTAGC
121 GGTGAAGGAG CAGTGGAGAG CAAGGAGCGA GGGTTGTTCG ATTTCATGGG GAAGAAAGAA121 GGTGAAGGAG CAGTGGAGAG CAAGGAGCGA GGGTTGTTCG ATTTCATGGG GAAGAAAGAA
181 GAGGAGAAGC CTCAAGAGGA GGTGATCGTA ACCGAGTTTG AAAAGGTTAA TATCGAAGAG181 GAGGAGAAGC CTCAAGAGGA GGTGATCGTA ACCGAGTTTG AAAAGGTTAA TATCGAAGAG
241 ACAAAGGCAG AGGAAGAAGG TGAGGAGAAG AAGAAGCATG GTCTCCTGGA GAAGCTTCAC 301 CGATCAGATA GCAGCAGCTC CAGCTCTTCA AGCGACGAGG AAGAAGGTGA AGGAGAAGAG241 ACAAAGGCAG AGGAAGAAGG TGAGGAGAAG AAGAAGCATG GTCTCCTGGA GAAGCTTCAC 301 CGATCAGATA GCAGCAGCTC CAGCTCTTCA AGCGACGAGG AAGAAGGTGA AGGAGAAGAG
361 AAGAAGAAGA AGAAAAAGAA GGAGAAGAAG GGACTGGAAG AAAAGATTGA AGAGAAATTA361 AAGAAGAAGA AGAAAAAGAA GGAGAAGAAG GGACTGGAAG AAAAGATTGA AGAGAAATTA
421 GAAGGGGAAA AGAAAGAAGA GGACACCTCA GTTCCAGTAG AGAAGTGCGA TGAGCCAGTA421 GAAGGGGAAA AGAAAGAAGA GGACACCTCA GTTCCAGTAG AGAAGTGCGA TGAGCCAGTA
481 GTCCAGCCAG AGACGCCAGA GAAGAAAGGT TTCCTCGAGA AGATCAAGGA GAAACTCCCT481 GTCCAGCCAG AGACGCCAGA GAAGAAAGGT TTCCTCGAGA AGATCAAGGA GAAACTCCCT
541 GGACAACACA AGAAAGCTGA AGAGGCCAGT AGCCCGGCTC CAGCCCCAGC AGCTGAGCCC 601 CATCACGAAG AGGAGTCAAA GGAAAAGAAG GGAATTTTGG AGAAAATCAA GGAGAAGCTT541 GGACAACACA AGAAAGCTGA AGAGGCCAGT AGCCCGGCTC CAGCCCCAGC AGCTGAGCCC 601 CATCACGAAG AGGAGTCAAA GGAAAAGAAG GGAATTTTGG AGAAAATCAA GGAGAAGCTT
661 CCTGGTTACC ACTCCAAATC AGATGAAGAA AAAGAAAAGG CTTGAGATTC AAGTTCACAT661 CCTGGTTACC ACTCCAAATC AGATGAAGAA AAAGAAAAGG CTTGAGATTC AAGTTCACAT
721 AAAAATACTG ATGGGATTGT GTGTTTCTTA AATTGTTCAA AAATTATCTG TTTTTGTTTT721 AAAAATACTG ATGGGATTGT GTGTTTCTTA AATTGTTCAA AAATTATCTG TTTTTGTTTT
781 TGAGGTTGTA TTTGTTGTAT ATTTCATATT TAAGTTTCTT CTTTTCCCAG CATTTTTAGT781 TGAGGTTGTA TTTGTTGTAT ATTTCATATT TAAGTTTCTT CTTTTCCCAG CATTTTTAGT
841 ACAAATCATA AGGAAAAAAA AAAGTTTGCT TT 841 ACAAATCATA AGGAAAAAAA AAAGTTTGCT TT
根据 SEQ ID NO: 17序列设计一对引物如下：  A pair of primers were designed according to the sequence of SEQ ID NO: 17 as follows:
Ghdehydrin09 ： SEQ ID NO: 7：  Ghdehydrin09 : SEQ ID NO: 7:
ATGGCCGAGGAGCATACCAGC  ATGGCCGAGGAGCATACCAGC
Ghdehydrin09R: SEQ ID NO: 8：  Ghdehydrin09R: SEQ ID NO: 8:
TCAAGCCTTTTCTTTTTCTTCATC  TCAAGCCTTTTCTTTTTCTTCATC
通过 SEQ ID NO:7和 SEQ ID NO: 8来克隆 Ghdehydrin09全长编码序列。 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以棉花的 cDNA为模板进行 PCR反应。 50 μΐ PCR反应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ cDNA， 1.0 μΐ PrimeSTAR^ 10 μΜ的引物 SEQ ID NO: 7和 SEQ ID NO: 8各 2.0 μ1， 以及 30 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 2min)， 72 °C 延伸 10 min。  The full-length coding sequence of Ghdehydrin09 was cloned by SEQ ID NO: 7 and SEQ ID NO: 8. The PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR^ 10 μΜ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 μl, and 30 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 2 min), extension at 72 °C for 10 min.
PCR扩增产物加 A尾： PCR产物加 2.5倍的无水乙醇， -20°C放置 10分钟， 离心，去上清，晾干，用 21 μΐ双蒸水溶解。加入 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μΐ 5 mM 的 dATP ， l ^ Ex Taq。反应条件： 70°C反应 30分钟。将得到约 600 bp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 Ghdehydrin09-pGEM质粒） ，然后将其转化到大肠杆菌 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 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。 SEQ ID NO: 7与 SEQ ID NO: 8进行菌液 PCR扩增 （反应体系及反应条件同上） ， 得到 4个阳性克隆， 送至英潍捷基 （上海） 贸易有限公司测序,所得序列为 SEQ ID NO: 2， 其编码 的 dehydrin09蛋白的氨基酸序列如 SEQ ID NO: 1所示。 The PCR amplification product was added with A tail: The PCR product was added with 2.5 times of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, and the supernatant was removed, dried, and dissolved in 21 μl of double distilled water. Add 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μΐ 5 mM dATP, l ^ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. Approximately 600 bp of DNA fragment was recovered (Omega Recovery Kit) and ligated into the pGEM T-easy vector (obtained Ghdehydrin09-pGEM plasmid), which was then transformed into E. coli JM109 competent cells and screened on LB solid medium containing 50 g/mL ampicillin (method supra). 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. SEQ ID NO: 7 and SEQ ID NO: 8 were used for PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the obtained sequence was SEQ. ID NO: 2, the amino acid sequence of the encoded dehydrin09 protein is shown in SEQ ID NO: 1.
dehydrin09蛋白的氨基酸序列： SEQ ID NO: 1 Amino acid sequence of dehydrin09 protein: SEQ ID NO: 1
1 MAEEHTSKAP ELESNVSGEG  1 MAEEHTSKAP ELESNVSGEG
21 AVESKERGLF DFMGKKEEEK  21 AVESKERGLF DFMGKKEEEK
41 PQEEVIVTEF EKVNIEETKA  41 PQEEVIVTEF EKVNIEETKA
61 EEEGEEKKKH GLLEKLHRSD  61 EEEGEEKKKH GLLEKLHRSD
81 SSSSSSSSDE EEGEGEEKKK  81 SSSSSSSSDE EEGEGEEKKK
101 KKKKEKKGLE EKIEEKLEGE  101 KKKKEKKGLE EKIEEKLEGE
121 KKEEDTSVPV EKCDEPVVQP  121 KKEEDTSVPV EKCDEPVVQP
141 ETPEKKGFLE KIKEKLPGQH  141 ETPEKKGFLE KIKEKLPGQH
161 KKAEEASSPA PAPAAEPHHE  161 KKAEEASSPA PAPAAEPHHE
181 EESKEKKGIL EKIKEKLPGY  181 EESKEKKGIL EKIKEKLPGY
201 HSKSDEEKEK A*  201 HSKSDEEKEK A*
Ghdehydrin09编码基因的核苷酸序列： SEQ ID NO: 2 Nucleotide sequence of the gene encoding Ghdehydrin09: SEQ ID NO: 2
1 ATGGCCGAGG AGCATACCAG CAAGGCCCCT GAGTTGGAGA GCAACGTTAG CGGTGAAGGA 1 ATGGCCGAGG AGCATACCAG CAAGGCCCCT GAGTTGGAGA GCAACGTTAG CGGTGAAGGA
61 GCAGTGGAGA GCAAGGAGCG AGGGTTGTTC GATTTCATGG GGAAGAAAGA AGAGGAGAAG61 GCAGTGGAGA GCAAGGAGCG AGGGTTGTTC GATTTCATGG GGAAGAAAGA AGAGGAGAAG
121 CCTCAAGAGG AGGTGATCGT AACCGAGTTT GAAAAGGTTA ATATCGAAGA GACAAAGGCA121 CCTCAAGAGG AGGTGATCGT AACCGAGTTT GAAAAGGTTA ATATCGAAGA GACAAAGGCA
181 GAGGAAGAAG GTGAGGAGAA GAAGAAGCAT GGTCTCCTGG AGAAGCTTCA CCGATCAGAT181 GAGGAAGAAG GTGAGGAGAA GAAGAAGCAT GGTCTCCTGG AGAAGCTTCA CCGATCAGAT
241 AGCAGCAGCT CCAGCTCTTC AAGCGACGAG GAAGAAGGTG AAGGAGAAGA GAAGAAGAAG241 AGCAGCAGCT CCAGCTCTTC AAGCGACGAG GAAGAAGGTG AAGGAGAAGA GAAGAAGAAG
301 AAGAAAAAGA AGGAGAAGAA GGGACTGGAA GAAAAGATTG AAGAGAAATT AGAAGGGGAA301 AAGAAAAAGA AGGAGAAGAA GGGACTGGAA GAAAAGATTG AAGAGAAATT AGAAGGGGAA
361 AAGAAAGAAG AGGACACCTC AGTTCCAGTA GAGAAGTGCG ATGAGCCAGT AGTCCAGCCA361 AAGAAAGAAG AGGACACCTC AGTTCCAGTA GAGAAGTGCG ATGAGCCAGT AGTCCAGCCA
421 GAGACGCCAG AGAAGAAAGG TTTCCTCGAG AAGATCAAGG AGAAACTCCC TGGACAACAC421 GAGACGCCAG AGAAGAAAGG TTTCCTCGAG AAGATCAAGG AGAAACTCCC TGGACAACAC
481 AAGAAAGCTG AAGAGGCCAG TAGCCCGGCT CCAGCCCCAG CAGCTGAGCC CCATCACGAA481 AAGAAAGCTG AAGAGGCCAG TAGCCCGGCT CCAGCCCCAG CAGCTGAGCC CCATCACGAA
541 GAGGAGTCAA AGGAAAAGAA GGGAATTTTG GAGAAAATCA AGGAGAAGCT TCCTGGTTAC541 GAGGAGTCAA AGGAAAAGAA GGGAATTTTG GAGAAAATCA AGGAGAAGCT TCCTGGTTAC
601 CACTCCAAAT CAGATGAAGA AAAAGAAAAG GCTTGA 601 CACTCCAAAT CAGATGAAGA AAAAGAAAAG GCTTGA
实施例 3 Ghdehydrin09基因植物表达载体构建 Example 3 Construction of Ghdehydrin09 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 ΡΤΠ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。 选择诱导型启动子 rd29A及 Tnos终止子分别作为 Gfrdefrydrm09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 Pnos promoter was used to replace the ΡΤΠ gene with a double enhancer. The 35S promoter reduces the expression of prion protein in plants. The inducible promoter rd29A and Tnos terminator were selected as promoters and terminators of the Gfrdefrydrm09 gene, respectively. The construction process is shown in Figure 1.
使用引物 SEQ ID NO: 9和 SEQ ID NO: 10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 ><PS Buffer、 3 μΐ 2.5 mM的 dNTP、 1.0 μ1 ΡΒΙ121、 1.0 μΐ Prime STAR、 10 μΜ的引物 SEQ ID NO:9 P SEQ ID NO: 10 各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 ( 94°C 变性 30 s， 56°C退火 30 s， 72 °C 延伸 30 s)， 72 °C 延伸 10 min。 通过 EcoRI、 Bglll酶切后将所得 PCR产物连接到 pCAMBIA2300 (Promega, T4 连 接酶盒）获得 pCAMBIA2300-l。  Using primers SEQ ID NO: 9 and SEQ ID NO: 10, Pnos was amplified using the plant expression vector PBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 μΐ PCR reaction system: 10 μΐ 5 ><PS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μ1 ΡΒΙ121, 1.0 μΐ Prime STAR, 10 μΜ primer SEQ ID NO: 9 P SEQ ID NO: 10 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 56 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min. The resulting PCR product was digested with EcoRI and Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase cassette) to obtain pCAMBIA2300-1.
SEQ ID NO: 9 ：  SEQ ID NO: 9:
GCkCGAA rraVTACAAATGGACGAACGGAT  GCkCGAA rraVTACAAATGGACGAACGGAT
SEQ ID NO: 10：  SEQ ID NO: 10:
A CCAGA 7tTAGATCCGGTGCAGATTATTTG  A CCAGA 7tTAGATCCGGTGCAGATTATTTG
使用引物 SEQ ID NO: 11和 SEQ ID NO: 12以 PBI121为模板扩增 Tnos,采 用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 1.0 μΐ PBI121 , 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 11和 SEQ ID NO: 12各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条 件： 94°C预变性 5 min， 33个循环 （94°C 变性 30 s， 58°C退火 30 s， 72 °C 延 伸 30 s)， 72 °C 延伸 10 min。 通过 Sacl、 EcoRI酶切后将所得 PCR产物连接 至 lj pCAMBIA2300-l (Promega, T4连接酶盒）获得 pCAMBIA2300-2  Primers SEQ ID NO: 11 and SEQ ID NO: 12 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 PrimeSTAR, 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 μl, and 31 μΐ Double distilled water. PCR 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. After digestion with Sacl and EcoRI, the obtained PCR product was ligated into lj pCAMBIA2300-l (Promega, T4 ligase cassette) to obtain pCAMBIA2300-2.
SEQ ID NO: 11：  SEQ ID NO: 11:
AAGdCTTGAATTTCCCCGATCGTTCAAA  AAGdCTTGAATTTCCCCGATCGTTCAAA
SEQ ID NO: 12： SEQ ID NO: 12:
CAGAA mCCAGTGAATTCCCGATCTAGTA  CAGAA mCCAGTGAATTCCCGATCTAGTA
使用引物 SEQ ID NO: 13和 SEQ ID NO: 14以拟南芥（哥伦比亚型 ， 购自 www.arabidopsis.org) 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 PCR反应体系： 10 μΐ 5 PS Buffer, 3 μΐ 2.5 mM的 dNTP， 1.0 μΐ拟 南芥 DNA， 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 13和 SEQ ID NO: 14 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 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 Arabidopsis rd29A promoter was amplified using Arabidopsis thaliana (Columbia type, available from www.arabidopsis.org) DNA using primers SEQ ID NO: 13 and SEQ ID NO: 14 (see Zeng J., et L. 2002). , Preparation of total DNA from "recalcit rant plant taxa", Acta Bot. Sin., 44(6): The method in 694-697 extracts Arabidopsis DNA). PrimeSTAR HS DNA polymerase using TaKaRa. 50 μΐ PCR reaction system: 10 μΐ 5 PS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ Arabidopsis DNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 13 and SEQ ID NO: 14 each 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min. The resulting PCR product was ligated by HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3
SEQ ID NO: 13：  SEQ ID NO: 13:
ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO: 14：  ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO: 14:
TGAd 6TCCAAAGATTTTTTTCTTTCCAATAG  TGAd 6TCCAAAGATTTTTTTCTTTCCAATAG
使用弓 I物 SEQ ID NO： 15和 SEQ ID NO: 16通过 PCR扩增 Ghdehydrin09 以 实施例 2所获得的阳性 Ghdehydrin09-pGEM质粒为模板）， 采用 TaKaRa 的 PrimeSTAR HS DNA聚合酶。50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer, 3 μ1 2.5 mM 的 dNTP， 1.0 μΐ Ghdehydrin09-pGEM质粒， 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 15和 SEQ ID NO: 16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 2min)， 72 °C 延伸 10 min。 通过 Pstl、 Sacl酶切后将所得 PCR产物连接到 (连接方法同上） pCAMBIA2300-3 ， 获得植物表达载体 rd29A- Ghdehydrin09-2300 (图 2)。  Using the SEQ ID NO: 15 and SEQ ID NO: 16 by PCR amplification of Ghdehydrin09 using the positive Ghdehydrin09-pGEM plasmid obtained in Example 2 as a template, TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μl 2.5 mM dNTP, 1.0 μΐ Ghdehydrin09-pGEM plasmid, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 15 and SEQ ID NO: 16 each 2.0 μl, and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min. After digestion with Pstl and Sacl, the obtained PCR product was ligated (ligation method as above) pCAMBIA2300-3 to obtain plant expression vector rd29A-Ghdehydrin09-2300 (Fig. 2).
SEQ ID NO: 15：  SEQ ID NO: 15:
TGA Cf^ TGGCCGAGGAGCATACCAGC  TGA Cf^ TGGCCGAGGAGCATACCAGC
SEQ ID NO: 16：  SEQ ID NO: 16:
AAGdCTCTCAAGCCTTTTCTTTTTCTTCATC 实施例 4 rd29 A-Ghdehydrin09-2300表达载体转化农杆菌  AAGdCTCTCAAGCCTTTTCTTTTTCTTCATC Example 4 rd29 A-Ghdehydrin09-2300 Expression Vector Transformation Agrobacterium
农杆菌 LBA4404 (购自 Biovector Science Lab,Inc) 感受态制备： 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 °C培养 1至 2天。 挑取单菌落接种于 5 ml含 50 g/ml利福平和 50 g/ml 链霉素的 LB液体培养基中，28°C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活化后的种子菌液 （1 :20的比例） 接种于 100 ml含 50 g/ml利福平和 50 μ§/ιη1链霉素的 LB液体培养基中， 28°C摇动培养 2-2.5 h至 OD6(K)=0.8。冰浴菌液 10 min， 每隔 3 min摇匀一次， 令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) Competent preparation: Agrobacterium tumefaciens LBA4404 was spotted on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin, 28 °C Incubate for 1 to 2 days. Pick a single colony inoculated in 5 ml containing 50 g/ml rifampicin and 50 g/ml The LB liquid medium of streptomycin was shaken overnight (about 12-16 h) at 28 ° C until the OD 6 (K) value was 0.4, and a seed bacterial liquid was formed. 5 ml of activated seed broth (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 g/ml rifampicin and 50 μ § /ιη1 streptomycin, and cultured at 28 ° C with shaking 2 2.5 h to OD 6 (K) = 0.8. The ice bath solution was shaken for 10 min every 3 min to allow the bacteria to enter the dormant state evenly. Centrifuge at 4000 g for 10 min at 4 °C, discard the supernatant; add a certain amount of ice-cold 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 °C, collect the precipitate; pre-cool with ice 10 The glycerol was repeatedly washed 3-4 times; the bacterial pellet was resuspended by adding 10% glycerol pre-cooled in an appropriate amount of ice bath to prepare LBA4404 competent cells, which were dispensed at 40 μM/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 rd29A-Ghdehydrin09-2300质粒， 混匀后 冰浴约 10 min。 将冰浴后的所述感受态细胞和阳性 rd29A-Ghdehydrin09-2300 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 0.1cm 的电击杯， MicroPulser (购自 Bio-Rad) 的程序设置为 "Agr"， 电击一次 。 立即取出电击杯， 加入 1 ml 28 °C 预热的 LB培养基。 快速而轻柔地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。 实施例 5 利用农杆菌介导的转化法获得转基因烟草 Transformation of Agrobacterium: LBA4404 competent cells were thawed on ice, and 1 μΐ of the positive rd29A-Ghdehydrin09-2300 plasmid obtained in Example 3 was added to 40 μΐ of the competent cells, and the mixture was mixed and ice bathed for about 10 min. The mixture of the competent cells after ice bath and the positive rd29A-Ghdehydrin 09-2300 plasmid was transferred to an ice-cold electric shock cup with a micropipette, and tapped to bring the suspension to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from Bio-Rad) on the slide of the electric shock chamber and push the slide to place the electric shock cup on the base electrode of the electric shock chamber. Using a 0.1 cm electric shock cup, the program of MicroPulser (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. Mix the mixture quickly and gently 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 μΐ of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 μ § /ιη1 rifampicin, 50 μ § /ιη1 streptomycin, 50 μ § /ιη1 Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Obtaining Transgenic Tobacco by Agrobacterium-mediated Transformation
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) 30 s, 用灭菌双蒸水洗两次。 再用 0.1%升汞浸泡 8 min， 用灭菌双蒸水洗两次， 完成表面灭菌。 将表面灭菌的烟草种子置于 MS固体培 养基（含 18.78 mM KN03， 1.25 mM KH2P04, 20.6 mM H4N03, 1.5 mM MgS04， 3.0 mM CaCl2, 50 μΜ ΚΙ, 100 μΜ Η3ΒΟ3， 100 M MnSO4， 30 M ZnSO4， 1 μΜ Na2Mo04， 0.1 M CoCl2， 100 μΜ Να2ΕϋΤΑ, 100 M FeSO4， 7.4 g/L琼脂， 蔗 糖 30 g/L, 余量为水） 上， 于无菌条件下发芽， 制备无菌苗。 取无菌苗叶片剪 成 5 mmx5 mm大小的叶盘， 用处于对数生长期的实施例 4中所得的含表达载 体 rd29A-Ghdehydrin09-2300的农杆菌 LBA4404阳性转化克隆的菌液浸染叶盘 10 min, 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+1 mg/L细胞分裂素 （BA) +0.1 mg/L萘乙酸（NAA) +50 mg/L卡 那霉素 +500 mg/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+50 mg/L卡那霉素 +500 mg/L头孢霉素） 中培养 30天 左右，待根系发达后将小苗转入含有 500 mg/L头孢霉素的 MS培养基上进行编 号保存。 To soak tobacco seeds with 75% alcohol (National Tobacco Medium Term Bank, obtained by: Institute of Tobacco, Chinese Academy of Agricultural Sciences, Library No. I5A00660) 30 s, washed twice with sterile double distilled water. Soak it in 0.1% liters of mercury for 8 min, and wash it twice with sterile double distilled water to complete surface sterilization. Surface-sterilized tobacco seeds were placed in MS solid medium (containing 18.78 mM KN0 3 , 1.25 mM KH 2 P0 4 , 20.6 mM H 4 N0 3 , 1.5 mM MgS0 4 , 3.0 mM CaCl 2 , 50 μΜ ΚΙ, 100 μΜ Η 3 ΒΟ 3 , 100 M MnSO 4 , 30 M ZnSO 4 , 1 μΜ Na 2 Mo0 4 , 0.1 M CoCl 2 , 100 μΜ Να 2 ΕϋΤΑ, 100 M FeSO 4 , 7.4 g/L agar, sucrose 30 g/L, The remaining amount is water), and the seeds are germinated under aseptic conditions to prepare sterile seedlings. Take sterile seedling blade The leaf discs of 5 mm×5 mm size were inoculated with the Agrobacterium LBA4404 positive transformation clone containing the expression vector rd29A-Ghdehydrin 09-2300 obtained in Example 4 in the logarithmic growth phase for 10 min, and the bacterial solution was aspirated. , co-cultured for 2 days in dark conditions (MS medium). The leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (NAA) + 50 mg/L kanamycin + 500 mg/L cephalosporin). The cells were cultured for about 45 days under light conditions. After the buds were grown, they were transferred to rooting medium (MS+50 mg/L kanamycin + 500 mg/L cephalosporin) for about 30 days. The seedlings were transferred to MS medium containing 500 mg/L cephalosporin for number storage.
取获得的转基因烟草叶片，提取 DNA (同实施例 3中拟南芥 DNA提取方 法），用 SEQ ID NO: 7和 SEQ ID NO: 8 ( 50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μ1 2.5 mM的 dNTP， 2.0 μΐ DNA, 1.0 μΙ Εχ Τ , 10 μΜ的引物 SEQ ID NO: 9 禾口 SEQ ID NO: 10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 2 min)， 72 °C 延伸 10 min， 并将 PCR鉴定为阳性的植株进行编号 （TQ1- TQ20) 并保存。 实施例 6 过表达 Ghdehydrin09 TQ代转基因烟草的耐旱模拟实验及功能鉴 定 The obtained transgenic tobacco leaves were taken, DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and SEQ ID NO: 7 and SEQ ID NO: 8 (50 μΐ PCR reaction system: 5 μΐ ΙΟχΕχ Buffer, 3 μl 2.5 mM dNTP, 2.0 μΐ DNA, 1.0 μΙ Τ Τ , 10 μΜ primer SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 μl, and 35 μΐ double distilled water. PCR reaction conditions: 94 °C pre-denaturation 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min, and numbering of plants identified as positive by PCR (T Q 1 - T Q 20) and preserved. Example 6 Drought tolerance simulation experiment and functional identification of Ghdehydrin09 T Q transgenic tobacco
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 TQ1-TQ20转基因 烟草及对照烟草 （非转基因） 组培苗分别移栽至蛭石上， 25°C、 10 h光培养 /14 h 暗培养循环， 每 5天浇一次 1/2MS液体培养基， 壮苗培养 15天之后进行干旱胁迫 实验， 将转基因烟草和对照烟草干旱 14天 (不浇水）， 25 °C、 10 h光培养 /14 h暗培 养循环。 TQ代转基因植株的抗旱性鉴定表明， 对照植株都萎蔫严重， 而 TQ1、 T03、 Τ05、 Τ06、 Τ08、 Τ09、 Τ012、 Τ。15八个转基因植株能够正常生长， 表现出显著的 耐旱性 （参见图 3， 以 TQ6、 TQ9为例， 其余未示出）。 实施例 7 在转录水平上验证 G/¾<fe/z ¾& ^9基因的表达 分别取对照烟草、 不显著耐旱转基因烟草 TQ代植株、 显著耐旱转基因烟草 To代植株 （生长状况良好） 干旱 14天的叶片各 0.05 g， 用植物 RNA提取试剂 盒 （Invitrogen) 提取的总 RNA。 用 HITACHI公司的紫外分光光度计 U-2001 测定所得总 RNA在 260 nm和 280 nm的吸光度值， 计算各个 RNA浓度。 依照 Invitrogen反转录试齐 Ll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: 8)。通过 SEQ ID NO:7和 SEQ ID NO: 8扩增 Ghdehydrin09， 检测 dehydrin09蛋白相对表达情况。 The sterilized vermiculite was soaked in 1/2 MS liquid medium. The T Q 1-T Q 20 transgenic tobacco and the control tobacco (non-transgenic) tissue culture seedlings obtained in Example 5 were transplanted to vermiculite, respectively, at 25 ° C, 10 h light culture / 14 h dark culture cycle, every 5 days. A 1/2 MS liquid medium was poured, and the drought stress test was carried out 15 days after the strong seedling culture. The transgenic tobacco and the control tobacco were dried for 14 days (without watering), 25 ° C, 10 h light culture / 14 h dark culture cycle. The drought resistance of the T Q transgenic plants showed that the control plants were severely wilted, while T Q 1 , T 0 3, Τ 0 5, Τ 0 6 , Τ 0 8 , Τ 0 9 , Τ 0 12, Τ. Fifteen transgenic plants were able to grow normally and showed significant drought tolerance (see Figure 3, taking T Q 6, T Q 9 as an example, the rest not shown). Example 7 The expression of the G/3⁄4<fe/z 3⁄4& ^9 gene was verified at the transcriptional level. Control tobacco, non-significant drought tolerant transgenic tobacco T Q plants, and significantly drought tolerant transgenic tobacco To plants (growth in good condition) Total RNA extracted by plant RNA extraction kit (Invitrogen) with 0.05 g of leaves per day for 14 days. The absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the respective RNA concentrations. according to Invitrogen reverse transcription assay Ll box Superscript III Reverse Transcriptase was used for reverse transcription (2 μg total RNA as template, reverse transcription primer SEQ ID NO: 8). The relative expression of dehydrin09 protein was detected by amplifying Ghdehydrin09 by SEQ ID NO: 7 and SEQ ID NO: 8.
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以上述反转录所得的 cDNA 为模板进行 PCR反应。 50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM的 dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 7和 SEQ ID NO: 8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°C 延伸 lmin)， 72 °C 延伸 10 min。  PCR was carried out using TaKaRa's PrimeSTAR HS DNA polymerase using the cDNA obtained by the above reverse transcription as a template. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 μl, and 30 μΐ Double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 29 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
产物电泳结果如图 4所示： M为 DNA Ladder Marker (DL2000,购自深圳瑞 真生物技术有限公司）， 1-3 为非转基因对照烟草， 4-6 为耐旱效果不显著的转 基因烟草 TQ代植株， 7-14为耐旱效果显著的转基因烟草 TQ代植株 （依次为： Τ01、 Τ。3、 Τ。5、 Τ。6、 Τ。8、 T09、 T012、 T015 )。图中所示条带大小与 Ghdehydrin09 的大小一致（约 600bp)。 结果表明， 对照烟草中没有外源 G/¾¾/z ¾/n 7i^基因转 录的信号， 耐旱效果显著的转基因烟草 TQ代植株中外源 G/^e/y^r w^基因的转 录较强， 耐旱效果不显著的转基因烟草 TQ代植株转录很弱或者没有转录。 The electrophoresis results of the product are shown in Figure 4: M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-3 is non-transgenic control tobacco, and 4-6 is transgenic tobacco T with insignificant drought tolerance. Q- generation plants, 7-14 are transgenic tobacco T Q plants with significant drought tolerance (in order: Τ 0 1, Τ. 3, Τ. 5, Τ. 6, Τ. 8, T 0 9, T 0 12 , T 0 15 ). The size of the band shown is consistent with the size of Ghdehydrin09 (approximately 600 bp). The results showed that there was no signal from the exogenous G/3⁄4⁄4/z 3⁄4/n 7i^ gene in the control tobacco, and the transcription of exogenous G/^e/y^rw^ gene in transgenic tobacco T Q plants with significant drought tolerance was compared. The transgenic tobacco T Q plants with strong, drought-tolerant effects were not strongly transcribed or transcribed.

Claims

权 利 要 求 书 Claim
1. 棉花的一个脱水素蛋白， 其序列为 SEQ ID N0: 1。 A cotton dehydrin protein having the sequence SEQ ID NO: 1.
2. 编码权利要求 1的脱水素蛋白的基因， 其序列为 SEQ ID NO: 2。  2. A gene encoding the dehydrin protein of claim 1, the sequence of which is 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 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 rd29A-Ghdehydrin09-2300载 体。  4. The vector of claim 3 which is the rd29A-Ghdehydrin 09-2300 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， 所述植物是烟草。  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 tobacco.
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， 其中所述植物是烟草。  8. The method of claim 7 wherein the plant is tobacco.
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， 其中所述植物是烟草。  10. The use of claim 9, wherein the plant is tobacco.
PCT/CN2013/076334 2013-05-28 2013-05-28 Cotton dehydrin protein, coding gene of same, and application thereof WO2014190489A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201380074552.0A CN105051059B (en) 2013-05-28 2013-05-28 A kind of cotton dehydrin protein and its encoding gene and application
PCT/CN2013/076334 WO2014190489A1 (en) 2013-05-28 2013-05-28 Cotton dehydrin protein, coding gene of same, and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2013/076334 WO2014190489A1 (en) 2013-05-28 2013-05-28 Cotton dehydrin protein, coding gene of same, and application thereof

Publications (1)

Publication Number Publication Date
WO2014190489A1 true WO2014190489A1 (en) 2014-12-04

Family

ID=51987850

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/076334 WO2014190489A1 (en) 2013-05-28 2013-05-28 Cotton dehydrin protein, coding gene of same, and application thereof

Country Status (2)

Country Link
CN (1) CN105051059B (en)
WO (1) WO2014190489A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005122697A2 (en) * 2004-06-21 2005-12-29 Carmel-Haifa University Economic Corp. Ltd Transgenic plants containing a dehydrin gene
CN101955521A (en) * 2010-09-21 2011-01-26 中国农业大学 Plant stress tolerance associated protein, and coded genes and application thereof
CN102080088A (en) * 2009-11-27 2011-06-01 创世纪转基因技术有限公司 Cotton dehydrin similar gene and application thereof
CN102492030A (en) * 2011-12-06 2012-06-13 吉林大学 Gene comprising dehydrin functional domain and application thereof in anti-drought alkali-resistant gene engineering

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005122697A2 (en) * 2004-06-21 2005-12-29 Carmel-Haifa University Economic Corp. Ltd Transgenic plants containing a dehydrin gene
CN102080088A (en) * 2009-11-27 2011-06-01 创世纪转基因技术有限公司 Cotton dehydrin similar gene and application thereof
CN101955521A (en) * 2010-09-21 2011-01-26 中国农业大学 Plant stress tolerance associated protein, and coded genes and application thereof
CN102492030A (en) * 2011-12-06 2012-06-13 吉林大学 Gene comprising dehydrin functional domain and application thereof in anti-drought alkali-resistant gene engineering

Also Published As

Publication number Publication date
CN105051059A (en) 2015-11-11
CN105051059B (en) 2018-03-16

Similar Documents

Publication Publication Date Title
WO2014172852A1 (en) Myb transcription factor myb1-1 of cotton and coding gene, and use thereof
CN112391406B (en) Method for promoting growth of strawberries and biological material used by same
WO2015042742A1 (en) Calcineurin b-like protein cbl-3 from cotton, and coding gene and use thereof
WO2014172847A1 (en) Myb transcription factor myb1-2 of thellungiella salsuginea and coding gene, and use thereof
WO2015042740A1 (en) Thellungiella halophila calcineurin b-like protein cbl-4, coding gene of same, and application thereof
WO2014169472A1 (en) Cotton leucine zipper protein bzip-3, coding genes of same, and application thereof
WO2014026312A1 (en) Cotton isopentenyl-transferase, gene encoding same and uses thereof
WO2014190489A1 (en) Cotton dehydrin protein, coding gene of same, and application thereof
WO2014082280A1 (en) Cotton zinc finger protein (czf4) and coding gene and application thereof
WO2014172842A1 (en) Cysteine protease cysp1-2 of cotton and coding gene, and use thereof
WO2014101153A1 (en) Cotton isopentenyl transferase ipt2, gene for encoding same, and application thereof
WO2015042749A1 (en) Thellungiella halophila dehydrin protein dh5, coding gene of same, and application thereof
WO2014101157A1 (en) Cotton isopentenyl transferase (ipt3), encoding gene and use thereof
WO2014082285A1 (en) Cotton zinc finger protein (czf5) and coding gene and use thereof
WO2014063269A1 (en) Cotton casein kinase, and coding gene and application thereof
WO2014107863A1 (en) Cotton homeotic-leucine zipper protein hdbzip-1 and coding gene and use thereof
WO2014172840A1 (en) Cysteine protease cysp1-1 of cotton and coding gene, and use thereof
WO2015042738A1 (en) Thellungiella halophila homeotic-leucine zipper protein hdbzip-3, coding gene of same, and application thereof
WO2014026311A1 (en) Cotton protein kinase and encoded gene and use thereof
WO2013181832A1 (en) Cotton avp1 protein and coding gene and application thereof
WO2013185257A1 (en) Dreb1-like transcription factor of cotton and coding gene and application thereof
WO2013185256A1 (en) Dreb1-like transcription factor of cotton and coding gene and application thereof
WO2014172825A1 (en) Tonoplast sodium-hydrogen anti-protein nhx2 of thellungiella halophila and coding gene and use thereof
WO2014190532A1 (en) Cotton pp2ac-type protein phosphatase pp2ac-4, coding gene of same, and application thereof
WO2014190453A1 (en) Cotton pp2ac-type protein phosphatase pp2ac-7, coding gene of same, and application thereof

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201380074552.0

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13885528

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13885528

Country of ref document: EP

Kind code of ref document: A1