WO2013123625A1 - 枣树谷胱甘肽过氧化物酶基因 - Google Patents

枣树谷胱甘肽过氧化物酶基因 Download PDF

Info

Publication number
WO2013123625A1
WO2013123625A1 PCT/CN2012/000306 CN2012000306W WO2013123625A1 WO 2013123625 A1 WO2013123625 A1 WO 2013123625A1 CN 2012000306 W CN2012000306 W CN 2012000306W WO 2013123625 A1 WO2013123625 A1 WO 2013123625A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
jujube
pcr
glutathione peroxidase
pgex
Prior art date
Application number
PCT/CN2012/000306
Other languages
English (en)
French (fr)
Inventor
曹秋芬
孟玉平
张晓娟
张春芬
曹尚银
薛华柏
Original Assignee
山西省农业科学院生物技术研究中心
山西维民生科技有限公司
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 山西省农业科学院生物技术研究中心, 山西维民生科技有限公司 filed Critical 山西省农业科学院生物技术研究中心
Publication of WO2013123625A1 publication Critical patent/WO2013123625A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01009—Glutathione peroxidase (1.11.1.9)

Definitions

  • the invention relates to the technical field of genetic engineering, in particular to a jujube glutathione peroxidase gene.
  • GPX which has antioxidant activity in animal cells, is thought to protect against hemolysis caused by red blood cell oxidation and is named glutathione peroxidase. In the past, this was considered to be the classic GPX. The enzyme with this function is now named GPX-1. Later, the Flohe research team purified and analyzed the distribution in cells and applied to various organic peroxides and H 2 0 2 GPX, studies have shown that it is a tetramer composed of four protein subunits, each containing one selenocysteine, and it is formed by the oxidation of enzymes in the form of ionized selenol Restore the active center.
  • GPXs have high catalytic activity and can rapidly remove excess reactive oxygen species (ROS) produced in the body, and most of GPXs use glutathione GSH as a substrate to catalyze the decomposition of organisms. H 2 0 2 .
  • ROS reactive oxygen species
  • GPXs containing selenocysteine residues in animals have strong catalytic activity, while most of the plants are cysteine residues, which greatly reduces the catalysis of GPXs in plants.
  • cysteine residues which greatly reduces the catalysis of GPXs in plants.
  • some people have studied the GPXs of citrus, replacing the cysteine residues with selenocysteine residues, but there is no activity similar to mammalian GPXs.
  • GPXs The structure of GPXs in plants, the specificity of substrates and the distribution of plant tissues are different. They play an important role in resisting external stress, but our understanding of their functions and structural characteristics is still limited. A lot of work needs to be done. Therefore, cloning and identifying GPXs in plants, studying the stress resistance mechanism and the molecular structure, expression, function and regulation of genes related to stress can provide some experimental data and lay a foundation for making full use of these excellent resistance genes to improve plant stress resistance. Theoretical basis.
  • An object of the present invention is to provide a jujube glutathione peroxidase gene which can be introduced into a plant as a gene of interest, to improve plant stress resistance, and to improve plant varieties.
  • the present invention is achieved by the following technical scheme: A jujube glutathione peroxidase gene, the nucleotide sequence of which is the sequence shown in SEQ ID NO: 1.
  • amino acid sequence encoded by the nucleotide sequence of the jujube glutathione peroxidase gene is a sequence as shown in SEQ ID NO: 2.
  • the protein encoded by the nucleotide sequence of the jujube glutathione peroxidase gene of the gene has an amino acid sequence as shown in SEQ ID NO: 2.
  • the jujube glutathione peroxidase gene is used for improving plant stress resistance.
  • the invention constructs the whole glutathione peroxidase gene of jujube tree from the cDNA library of Ziziph jujuba Mill hupingzao) from the horticultural crop biotechnology research office of Shanxi Agricultural Biotechnology Research Center. Sequence, name it glutathione Eero dase ). Bioinformatics analysis showed that the full-length cDNA sequence of the gene was 510 bp, encoding 170 amino acids, including 16 acidic amino acids and 19 basic amino acids. The relative molecular weight of the protein was calculated to be 19.26 KD, and the theoretical isoelectric point was 5.00. The fat index is 76.69.
  • the sequence of the downstream primer for GY2 is: 5'- ATCCCGGGTTGAGATTCCCAAGA-3', containing the Sma I restriction site (ie underlined portion), termination of two protective bases AT and glutathione peroxidase Codon TGA.
  • the restriction endonucleases EcoR I and Sma I were modified and ligated into the plant expression vector PEZR(K)-LNY, and then screened for transformants by PCR containing karnataka-containing LB plates, PCR identification, restriction enzyme digestion, and sequencing.
  • the plant expression vector PEZR K ⁇ -LNY-Z G was successfully constructed.
  • the plant expression vector carrying the target gene, PEZR(K)-LNY- was transferred into competent cells of Agrobacterium LBA4404 by freeze-thaw method, and the transformants were screened by LB plate containing kanamycin, identified by PCR, identified by enzyme digestion, and identified by enzyme digestion. Sequencing confirmed that the engineering strain PEZR Q-LNY-Z G was successfully constructed.
  • Agrobacterium tumefaciens-mediated gene transformation into Arabidopsis thaliana was screened by 1/2MS medium containing kanamycin to obtain the T Q generation of transgenic Arabidopsis plants containing the gene of interest.
  • the target gene was confirmed by PCR to be successfully transferred to Arabidopsis thaliana.
  • the 2-butoxy-generation seeds of Arabidopsis and the wild-type (WT) Arabidopsis Transgenic seeds were sown in 1 / 2MS medium with different concentrations of NaCl, transgenic plants and T 2 generation for evaluation of salt tolerance, was observed
  • the growth status of the Arabidopsis thaliana plants was significantly better than that of wild-type (WT) Arabidopsis thaliana plants under salt stress treatment.
  • Transgenic T 2 Arabidopsis plants and wild type (WT ) that will grow two true leaves Arabidopsis thaliana plants were treated with salt tolerance and drought tolerance.
  • the growth status of Arabidopsis thaliana was observed after 15 days. It was found that the salt tolerance of Arabidopsis thaliana with Z-GP gene was significantly improved, and the drought tolerance of transgenic Arabidopsis thaliana was significantly improved.
  • RNA of the capsicum seedlings was extracted by CTAB method.
  • the OD 26Q value and formaldehyde denaturing gel electrophoresis showed that the concentration and purity of the extracted RNA were in accordance with the standard and the quality was qualified.
  • the total RA of reverse transcription was analyzed by quantitative PCR, and it was confirmed that it could be induced by drought and NaCl stress.
  • the present invention constructs a recombinant prokaryotic expression vector pGEX-4T-2-ZjGPX, which is realized in E. coli.
  • Prokaryotic expression in BL21 (DE3) provides experimental data for the study of the structure, function and regulation of the protein encoded by this gene.
  • the present invention separates the jujube glutathione peroxidase gene ZjGPX from jujube tree for the first time, and the gene is introduced into the plant as a target gene, thereby improving plant stress resistance, and is important for plant variety improvement.
  • Practical significance, in-depth study of the stress resistance mechanism of plants with excellent resistance can help to clarify the molecular structure, expression, function and regulation of stress-related genes, in order to further make full use of these excellent resistance genes to improve plant resistance Sex provides certain experimental data and lays a theoretical foundation. It also helps to understand the role of GPXs in plant reactive oxygen species signaling and promotes the understanding of reactive oxygen species as signaling molecules.
  • Figure 1 is a dendrogram of amino acid homology with known genes.
  • Figure 2 is a three-dimensional structure prediction diagram of glutathione peroxidase of jujube.
  • Figure 3 is a map of the gene product of the PCR target.
  • Figure 4 shows the pGEX-4T-2 and the target gene restriction map.
  • Figure 5 is a diagram showing the restriction enzyme digestion of the recombinant plasmid.
  • Figure 6 is a PCR identification diagram of the engineered strain pGEX-4T-2-Z OP-B.
  • Figure 7 shows SDS-PAGE analysis of pGEX-4T-2-Z GP-B induced product.
  • Figure 8 shows the effect of IPTG on protein expression and purification of the fusion protein.
  • Figure 9 is a map of the gene product of the PCR target.
  • Figure 10 is a restriction map of PEZR(K)-LNY and the gene of interest.
  • Figure 11 is a restriction map of the recombinant plasmid.
  • Figure 12 shows the selected transgenic Z GP gene Arabidopsis thaliana T Q seedlings.
  • Figure 13 shows the selected transgenic Zj'G gene Arabidopsis thaliana T c plants.
  • Figure 14 shows Arabidopsis thaliana gene detection in different strains of transgenic ZG gene Arabidopsis thaliana.
  • Figure 15 shows the germination of transgenic Z GP gene Arabidopsis thaliana and WT Arabidopsis seeds on different concentrations of NaCl medium.
  • Figure 16 shows the growth of transgenic Arabidopsis thaliana and WT Arabidopsis plants on different concentrations of NaCl medium.
  • Figure 17 is a comparison of the growth of transgenic Arabidopsis thaliana and WT Arabidopsis plants after salt stress.
  • Figure 18 is a comparison of the growth of transgenic Arabidopsis thaliana and WT Arabidopsis plants after drought stress.
  • Figure 19 is an electrophoresis map of RNA formaldehyde denatured gel.
  • Figure 20 is a comparison table of Z G relative expression of capsicum seedlings after different concentrations of NaCl stress.
  • Figure 21 is a comparison table of relative expression levels of pepper jujube tissue culture seedlings after PEG-6000 stress (simulated drought treatment).
  • the invention constructs jujube glutathione peroxide from the cDNA library of the result branch (ie jujube hanging and falling branch) from the horticultural crop biotechnology research office of Shanxi Agricultural Biotechnology Research Center. The full sequence of the enzyme gene.
  • the determined gene sequence was searched by Blastx for NCBI nucleotide database for sequence similarity analysis; DNAStar was used for amino acid sequence alignment and the phylogenetic tree of the protein was used; ProtPamm was used to calculate the relative molecular weight and theoretical isoelectric point of the protein; Conservative sequence analysis was performed at http://www.expasy.org/prosite; the amino acid sequence of this protein was submitted to the Swiss-Model to predict its tertiary structure.
  • DNAStar constructs a phylogenetic tree.
  • the amino acid sequence of the glutathione peroxidase gene of jujube is closely related to the GPX of ramie, and the glutathione peroxidase of seven species is clustered into 5 Groups, Arabidopsis and poplar are grouped together; maize and sorghum are grouped together; jujube, grape, and ramie are grouped together; cluster analysis also indicates glutathione peroxidase and 6 species GPX
  • the amino acid homology reached 73.8% or more; the Zjd gene cDNA sequence was 510 bp in length.
  • ProtParam analyzed the sequence, which encoded 170 amino acids, including 16 acidic amino acids and 19 basic amino acids.
  • the relative molecular weight of the protein was calculated to be 19.26 KD, the theoretical isoelectric point was 5.00, and the fat index was 76.69.
  • glycopeptide peroxidase active site has an amino acid sequence of the 31st amino acid to the 46th amino acid "G vLLIvNVaSkCGmT” and a glutathione peroxidase conserved peptide having the amino acid sequence "LAFPCNQF"", from the 68th amino acid to the 75th amino acid; search for the 'OP homology model 2p5rA in the protein structure database, and homologous modeling with the Swiss Model program (http: au au.expasy.org/tools/) Homology modeling), speculatively, the three-dimensional structure of the ZG lY gene-encoded protein is shown in Figure 2.
  • the glutathione peroxidase of jujube has a very high similarity with the glutathione peroxidase of poplar, suggesting that it may Poplar glutathione peroxidase has a similar function.
  • the above bioinformatics analysis methods are all conventional techniques well known to those skilled in the art.
  • Escherichia coli DH5a strain cloning vector pSPORTl-ZjGPX, prokaryotic expression vector PGEX-4T-2 were preserved by the Horticultural Crop Research Laboratory of Biotechnology Research Center of Shanxi Academy of Agricultural Sciences. _ 1.2 Enzymes and kits
  • DNA recovery kit QIAquick® was purchased from QIAGEN; restriction endonucleases amH I , Sma I , Sal I , T4 DNA ligase, DNA Marker (15000 bp, 2000 bp ladder) were purchased from Dalian TaKaRa (Bao Bio) Engineering Co., Ltd. the company.
  • the antibiotic ampicillin was purchased from Shanghai Shenggong Bioengineering Service Co., Ltd. Plasmid extraction and other reagents used in agarose gel electrophoresis were purchased from Tianjin Tianda Chemical.
  • the main equipments are electronic balance (Sarorius BS200s-WEl), ultra-clean workbench (Shanghai Xunbo Medical Equipment Factory Vs-840-2), water-tight constant temperature incubator (Shanghai Yuejin Medical Devices No.
  • Escherichia coli E.co/ ⁇ DH5 (competent cells) were prepared by CaCl 2 method. For specific methods, see Molecular Molecular Biology Experiment. Guidelines, liquids and containers that come into contact with bacteria are sterilized.
  • the upstream primer G1 sequence of the glutathione peroxidase gene of jujube is:
  • 5'-TATGGATCCATGACTAGCCAGCCCA-3' contains the awH I restriction site (ie underlined), three protective bases TAT, and the start codon ATG of the glutathione peroxidase ZG gene .
  • the sequence of the downstream primer G2 is: 5' - ATCCCGGGTCATGAGATTCCCAAGA - 3', containing the S ⁇ m I restriction site (ie underlined), two protected bases AT, and glutathione peroxide The stop codon TCA of the enzyme gene.
  • the plasmid pGEX-4T-2 and pSPORTl-Z O were rapidly extracted by alkaline lysis. The specific steps are described in the Guide to Molecular Biology.
  • the cloning vector pSPORTl (carrying the target fragment) was used as a template, and PCR was amplified using the designed specific primers. Gradient PCR techniques were used to set different temperatures to find the optimum annealing temperature of 61 °C.
  • PCR reaction system 1 OxPCR buffer 5 ⁇ 1 (containing Mg 2+ ion), dNTP 1 ⁇ , 1 ⁇ 1 of each upstream and downstream primers (20 ⁇ >1/ ⁇ ), rTaq polymerase 0.3 ⁇ 1, template DNA ⁇ ⁇ ( 2.3 ⁇ / ⁇ 1 ), sterilized ultrapure water is added to a total volume of 50 ⁇ 1.
  • the amplification conditions were: 95 ⁇ pre-denaturation for 5 min, denaturation at 94 °C for 1 min, annealing at 61 °C for 1 min, extension at 72 °C for 2 min, a total of 40 cycles, and finally 72 ⁇ extension for 5 min.
  • the PCR product of the target gene Zj'GP and the vector pGEX-4T-2 were digested with i ⁇ H I and ⁇ ⁇ I, respectively.
  • the digestion reaction system of the PCR product of the target gene is as follows:
  • Total volume 50 ⁇ Note: The volume of the PCR product depends on its concentration, and at least 1 to 2 g of the target gene fragment is digested. Finally add ultrapure water to a total volume of 50 ⁇ 1.
  • the target gene and vector after double digestion were purified according to the instructions of the DNA purification kit.
  • the prepared pGEX-4T-2 vector was ligated with the ZGPJ3 ⁇ 4 gene fragment by ⁇ 4 DNA ligase, and the total binding reaction system was 10 ⁇ l, including 0.5 ⁇ DNA4 DNA ligase, 300 ng ZG 3 ⁇ 4 gene fragment and 300 ng pGEX-4T. -2, 16 °C overnight connection.
  • E. coli DH5 Competnt cells, mix gently, place on ice for 30 min. Place in a circulating water bath pre-warmed to 42 ° C for heat shock 90 s Quickly transfer the tube to the ice bath and let the cells cool for 3-5 minutes. Force OO ⁇ SOC medium, transfer the tube to the 37'C shaker, warm the bath for 40 minutes to resuscitate the bacteria, and gently shake the cells during the recovery period. Below 225 r/min) Transfer the appropriate volume of the transformed bacterial solution to an LB plate containing ampicillin and spread it evenly. Place the plate in the greenhouse until the liquid is absorbed, invert the plate, and incubate at 37 ° C overnight.
  • the transformed bacteria were grown on the LB plate containing ampicillin, and the transformed single colonies were picked up to 50 ⁇ l ultrapure water with a sterile toothpick, and the bacteria were lysed by boiling water for 5 min. ⁇ was used as a template for PCR positive identification.
  • the reaction system was: lOxPCR buffer 1.5 ⁇ 1 (containing Mg 2+ ), ⁇ 0.2 ⁇ 1, upstream and downstream primers 0.5 ⁇ (20pmol/L), rTaq polymerase ⁇ , ⁇ , template ⁇ ) ⁇ 1 ⁇ 1 (2.3 ⁇ ⁇ / ⁇ 1), sterilized ultrapure water plus 11.2 ⁇ 1 to a total volume of 15 ⁇ 1.
  • the amplification conditions are the same as before.
  • the transformant identified as positive by PCR was streak cultured. 2.7.2 enzyme digestion identification
  • the plasmid DNA was extracted by alkaline lysis, and the recombinant plasmid was digested with restriction endonucleases a H I and & / / 1 .
  • the PCR product and pGEX-4T-2 plasmid were digested with &TMHI and Sma I, and the digested product was subjected to agarose electrophoresis.
  • the PCR product size was about 510 bp, and the pGEX-4T-2 vector fragment was about 4.9 Kb.
  • the results were consistent (as shown in Figure 4, in the figure: Mi: DL2000; 1: target gene OP fan-cut product; M 2 : DL15000 Marker; 2: pGEX-4T-2 digested product).
  • the fragment of interest and the vector fragment were separately purified for ligation.
  • plasmid pGEX -4T-2-ZjGPX was identified by double digestion with ⁇ wzH I and the results are shown in Figure 5, in which: DL2000 Marker; 1-7: positive plasmid.
  • the size of the double-digested fragment was about 515 bp, which was consistent with the expected results. It was preliminarily concluded that the recombinant plasmid was successfully constructed, and the size of the recombinant plasmid was about 5.4 Kb.
  • Example 3 Expression of Z GPAT in Escherichia coli and Protein Purification 1 Materials, reagents and instruments
  • the instruments used in this embodiment mainly include ultrasonic instruments (West Germany Elma D-7700), DYY-6B electrophoresis apparatus (Beijing Liuyi Instrument Factory), DYCZ-28A type electrophoresis tank (Beijing Liuyi Instrument Factory), Sony digital camera (Sony (China) Co., Ltd. DSC-T500).
  • ultrasonic instruments West Germany Elma D-7700
  • DYY-6B electrophoresis apparatus Beijing Liuyi Instrument Factory
  • DYCZ-28A type electrophoresis tank Beijing Liuyi Instrument Factory
  • Sony digital camera Sany (China) Co., Ltd. DSC-T500.
  • Other instruments can be found in the Guide to Molecular Biology Experiments.
  • Reagent formulation for fusion protein purification extraction buffer: 140 mM NaCl; lOmM Na 2 HP0 4 ; 1.8 mM KH 2 P0 4 (pH 7.5).
  • the first buffer 0.1 M Tris-HCl; 0.5 M NaCl (pH 8.5).
  • the second buffer 0.1 M sodium acetate; 0.5 M NaCl (pH 4.5).
  • the third buffer 140 mM NaCl; lOmM 10 mM Na 2 HP0 4 ; 1.8 mM KH 2 P0 4 (pH 7.5).
  • E. coli BL21 (DE3) competent cells were prepared by CaCl 2 method. For specific methods, see the Guide to Molecular Biology. Dispense into ⁇ or 200 ⁇ 1 aliquots, freeze in liquid nitrogen, store at -70'C for later use.
  • the recombinant plasmid pGEX-4T-2-Z OPJ and the vector pGEX-4T-2 constructed in Example 2 were introduced into the competent cells of the expression strain BL21 (DE3) by heat shock method, and applied to an LB plate containing ampicillin. The culture was allowed to stand overnight at 37 °C.
  • IPTG was added to the culture at a final concentration of 1.0 mmol/L, and incubation was continued for 4 hours at 37 °C. .
  • the concentration of the gel is 70 ⁇ , but the current should not exceed 20 mA.
  • the bromophenol blue in the gel reaches the separation gel, it is boosted to 150 V and continues to electrophoresis.
  • bromophenol blue is electrophoresed to the bottom of the gel. (ie the bottom of the separation gel) 1 cm, the electrophoresis is over.
  • the positive engineering strain pGEX-4T-2-ZG-B was selected and cultured at 37 °C to OD 6 (W value was about 0.5, IPTG to final concentration was 1.0 mmol/L, induced culture at 37 °C). 2 mL of bacterial solution was taken at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, using pGEX-4T-2-B without insert as a blank control, control strain pGEX-4T-2- B also added IPTG to a final concentration of 10 mmol / L, 37 'C induced culture, 2 mL of bacterial solution was taken at 4 h, samples were prepared according to the above sample preparation method, each sample was taken 20 ⁇ 1, and SDS-PAGE was performed. To analyze the effect of IPTG on protein expression at different induction times.
  • the identified positive engineering strain pGEX-4T-2-Z OP-B was selected and cultured at 37 °C until the OD 6TO value was about 0.5.
  • IPTG was added to a final concentration of 0.1, 0.4, 0.8, 1.0, 1.2, and 1.6 mmol, respectively.
  • /L induction culture at 37 °C for 4 h, pGEX-4T-2-B without insert as a blank control, control PGEX-4T-2-B with IPTG to a final concentration of 1.0 Mmmol/L, samples were prepared according to the above sample preparation method, and each sample was taken for 20 ⁇ l, and subjected to SDS-PAGE to analyze the effect of IPTG on protein expression at different induced concentrations.
  • the engineering strain pGEX-4T-2-ZOP B was sampled by IPTG for 4 h, and the electrophoresis results are shown in Fig. 7.
  • M protein Marker
  • 13 control empty cells
  • 5, 6, 9, 10 expression Positive cells of the 45KD fusion protein.
  • E. coli BL21 (DE3) containing plasmid pGEX-4T-2-Zj'GP was at about 45KD compared with the control empty cell PGEX-4T-2-B under IPTG induction conditions.
  • a specific protein band was produced, indicating that the expression of glutathione peroxidase in jujube was induced. (GST size is about 26KD, the target protein size is 19.26KD, so the fusion protein size is about 45KD).
  • the artificial bacteria pGEX-4T-2-Z/GP-B with an OD 600 value of about 0.5 was added with IPTG to a final concentration of 1.0 mmol/L, and cultured at 37 °C for 1 h, 2 h, 3 respectively. 2 mL of bacterial solution was taken at h, 4h, 5 h, and 6 h.
  • IPTG was added to the fresh bacterial solution of engineering bacteria pGEX-4T-2- 'GP-B with an OD value of about 0.5 to a final concentration of 0.1, 0.4, 0.8, 1.0, 1.2, 1.6 mmol/L, respectively, and induced for 37 hours.
  • Bacteria Bacteria.
  • the bacterial solution was taken and subjected to SDS-PAGE electrophoresis.
  • the results of electrophoresis are shown in Figure 8.
  • the IPTG induction time was 5h
  • the expression of the target protein was the highest.
  • the final concentration of IPTG was 0.4 mmol/L
  • the expression of the target protein was the largest.
  • the fusion protein is expressed in a large amount, and the bacterial lysate is collected, and combined with the GST-tagged protein purification resin by affinity chromatography, and purified by multiple elution to obtain a GST-ZGP fusion protein with a molecular weight of 45 KD, the first band, visible
  • the GST fusion protein has a high affinity efficiency with the GST-tagged protein purification resin. See Fig.
  • M protein Marker
  • 1 purified GST-ZG/Y fusion protein
  • 2 control GST protein expression
  • 3-8 induction of GST-Zj'G fusion protein expression at different times, induction time is Lh, 2h, 3h, 4h, 5h, 6h
  • 9-14 Different concentrations of GST-ZOP fusion protein were induced, IPTG to final concentrations were 0.1, 0.4, 0.8, 1.0, 1.2, 1.6mmol/Lc
  • Plant expression vector PEZR(K)-LNY (containing yellow fluorescent protein (YFP) marker gene), Escherichia coli (co//) DH5a strain, and recombinant plasmid pSPORTl-ZjGPX containing Z'G3 ⁇ 4f gene were all biotechnology of Shanxi Academy of Agricultural Sciences. The Research Center for Horticultural Crops is kept.
  • DNA recovery kit TaKaRa DNA Fragment Purification Kit Ver.2.0, Restriction enzyme "HI, Smal, EcoR I ⁇ HindllL T4 DNA ligase (DNA Ligation Kit Ver.2.0), DNA Marker, etc. were purchased from Dalian TaKaRa ( ⁇ ) Engineering Co., Ltd.
  • Phenol Chloroform: isoamyl alcohol (V: V: V, 25: 24: 1 )
  • TE solution (10 mM Tris-Hcl, ImM EDTA, pH 8.0)
  • the upstream primer is GY1: 5'-ACGAATTCTCATGACTAGCCAGC-3', which contains the £co H restriction site (ie underlined), three protected bases ACG and the start codon ATG of this protein.
  • the downstream primer is GY2: 5'- ATCCCGGGTTGAGATTCCCAAGA-3', containing the Sma I restriction site (ie underlined), two protective bases AT and the stop codon TGA of glutathione peroxidase .
  • GY2 5'- ATCCCGGGTTGAGATTCCCAAGA-3', containing the Sma I restriction site (ie underlined), two protective bases AT and the stop codon TGA of glutathione peroxidase .
  • the DH5a bacterial solution of the pSPORTl-Z G/ 3 ⁇ 4r plasmid was cultured overnight at 37 ° C with 2 mL of LB liquid medium plus ⁇ ampicillin (100 mg/mL).
  • the DH5a broth containing the PEZR(K)-LNY plasmid was cultured in 2 mL of LB liquid medium plus ⁇ kanalin (100 mg/mL) and cultured overnight.
  • the dried plasmid was dissolved in 20 ⁇ M TE and stored at -20 °C until use.
  • the cloning vector pSPORTl (carrying the target fragment) was used as a template, and PCR was amplified using the designed specific primers. Gradient PCR techniques were used to set different temperatures to find the optimum annealing temperature of 52 ⁇ .
  • PCR reaction system 1 OxPCR buffer 5 ⁇ 1 (containing Mg 2+ ions), dNTP 1 ⁇ , 1 ⁇ 20pmol/L for each of the upstream and downstream primers, rTaq polymerase 0.3 ⁇ 1, template DNA ⁇ ⁇ (2.3 ⁇ / ⁇ 1), The bacteria ultrapure water is 40.7 ⁇ 1, and the total volume is 50 ⁇ 1.
  • the amplification conditions were: pre-denaturation at 95 °C for 5 min, denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, extension at 72 °C for 2 min, 40 cycles, and finally extension at 72 °C for 5 min.
  • Both the PCR product of the target gene and the multiple cloning site of the vector PEZR(K)-LN Y have EcoR I and Sma I restriction endonuclease sites, and the target gene Zj'G / ⁇ is digested with EcoR I and I, respectively.
  • the PCR product and the carrier PEZR(K)-LNY, which have the same sticky ends, are ready for ligation.
  • the digestion reaction system of the target gene ZG 3 ⁇ 4 PCR product is as follows:
  • the enzymatic cleavage reaction system of the carrier PEZR(K)-LNY is as follows:
  • the target gene and vector after double digestion were purified according to the instructions of the DNA purification kit.
  • the target gene fragment and vector PEZR(K)-LNY were digested with EcoR I and Sma I, and then purified according to the instructions of the agarose gel DNA recovery kit TaKaRa DNA Fragment Purification Kit Ver.
  • the purified PEZR (K LNY vector was ligated with the fragment by T4 DNA ligase.
  • the DNA fragment of the plasmid vector PEZR(K)-LNY was mixed with the DNA fragment of the target gene Z'OP to prepare a DNA having a volume of about 5 ⁇ l.
  • the solution (the molar ratio of the carrier DNA to the inserted DNA is generally: 0.03 pmol: 0.1 to 0.3 pmol), and an equal volume of Solution I in the DNA Ligation Kit is added to the above DNA solution, and thoroughly mixed.
  • the total volume of the reaction system is After 10 ⁇ , mix thoroughly and react at 16 ° C overnight.
  • the transformed bacteria grew out on the LB plate containing kanamycin resistance, and the transformed single colonies were picked up into 50 ⁇ l ultrapure water with a sterile toothpick, and the bacteria were lysed by boiling water for 5 min. ⁇ was used as a template for PCR positive identification.
  • the reaction system was: lOxPCR buffer 1.5 ⁇ 1 (containing Mg 2+ ), ⁇ 0.2 ⁇ 1, upstream and downstream primers 0.5 ⁇ 1 (20pmol/L), rTaq polymerase ⁇ . ⁇ , template DNA ⁇ (2.3 ⁇ 1), sterilized ultrapure water plus 11.2 ⁇ 1 to a total volume of 15 ⁇ 1.
  • the amplification conditions are the same as before.
  • the transformant identified as positive by PCR was streak cultured.
  • the transformant which was identified as positive by PCR was extracted with plasmid plasmid by alkaline lysis method, and the recombinant plasmid was digested with HwHII and Sa HI by restriction endonuclease, and the result of electrophoresis was observed, and the positive strain was sent to the often-finished gene company for sequencing. Verify the correctness of the reading frame of the recombinant plasmid.
  • the PCR product of G3 ⁇ 4 and the PEZR(K)-LNY expression vector were digested with I and >3 ⁇ 4 ⁇ I, and the digested product was digested. Agarose gel electrophoresis, the results are shown in Figure 10, in the figure, M : DL15000 arker ; 1: target gene digestion product; 2: PEZR (K)-LNY digestion product, PCR product size is about 510 bp, PEZR The (K)-LNY vector fragment was approximately 11.7 Kb, respectively, consistent with the expected results.
  • the target fragment and the vector fragment were purified according to the kit instructions for ligation.
  • the ligation product was transformed into E. coli DH5a, and a positive single colony was picked from the medium containing kanamycin for PCR identification.
  • the plasmid DNA of the positive clone was extracted and digested with H III and awH I to generate a band of about 11.7 Kb (as shown in Fig. 11, in the figure, M: DL15000 Marker; 1-10: positive plasmid) ), indicating that the target has been included in the vector.
  • sequencing results also indicated that the sequence of the exogenous insert of the recombinant plasmid was correct.
  • the above results confirmed that the Z GP gene has been successfully constructed on the plant expression vector PEZR(K)-LNY, and the vector was named
  • Wild-type Arabidopsis seeds were obtained from the Horticultural Crop Research Laboratory of the Biotechnology Research Center of Shanxi Academy of Agricultural Sciences.
  • the culture medium was purchased from the Shanxi Provincial Institute of Horticultural Crops.
  • Plant expression vector PEZR(K)-LNY-d and Agrobacterium tumefaciens LBA4404 were all preserved by the Horticultural Crop Research Laboratory of Shanxi Agricultural Biotechnology Research Center.
  • Kanamycin was purchased from Shanghai Shenggong Bioengineering Service Co., Ltd.; reagents used in ⁇ and 1/2 MS media were purchased from Tianjin Tianda Chemical.
  • Peptone 0.05 g / L peptone 0.05 g / L, yeast extract 0.01 g / L, sucrose 0.05 g / L, magnesium sulfate 0.005 g / L.
  • the solid medium should be added to a final concentration of 1.0 ° /. Agar powder.
  • Arabidopsis seeds were first soaked in 95% ethanol for 30 ⁇ 60s; then transferred to 2.65% sodium hypochlorite for 5min, washed upside down, centrifuged at 5000rpm for 2min; sterilized washed three times; then suspended in 0.1% agar powder sol The seed was spotted on a 1/2 MS solid medium with a pipette tip and sealed; after 4 days of dark vernalization at 4 °C, the Arabidopsis seeds were subjected to 2TC, 16 h / 8 h photoperiod conditions. Culture; When two leaves of Arabidopsis thaliana grow, they are transplanted into a nutrient substrate and cultured.
  • the recombinant plasmid PEZR(K)-LNY-Z G ⁇ was transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, and the plasmid DNA of the positive clone was extracted, and the recombinant plasmid was transferred to Agrobacterium by PCR.
  • the correct positive clones were selected and cultured in lOmLYEB liquid medium (containing a final concentration of 50 g/mL of Kana antibiotic). After overnight culture, the cells were collected by centrifugation at 6000 rpm for 5 min, and the cells were suspended in the medium to an OD 6 ⁇ M) value. It is about 0.8 and is used to transform Arabidopsis plants.
  • Arabidopsis plants When Arabidopsis plants form flower buds, they can be used for Agrobacterium transformation.
  • the transformation medium containing Agrobacterium was poured into a beaker, and the flower buds of the Arabidopsis plants were partially immersed in the transformation medium 3 ⁇ 5S, and the infiltrated plants were placed in a plastic tray, covered with a film, and placed in the dark for 12 hours. The membrane is then uncovered and the transformed Arabidopsis plants are continued to be cultured under normal conditions.
  • the pods of Arabidopsis thaliana are yellow and want to be cracked, the seeds are harvested. This is the T Q seed.
  • Transgenic Arabidopsis plants T c sterilized seeds were sown in 1 / 2MS medium (containing a final concentration of 50 g / mL kanamycin in the dark 4 ° C after vernalization 2d disposed 22'C, 16h / 8h Cultured under photoperiod.
  • the seedlings of the transformed Arabidopsis thaliana grow normally, and the leaves of the untransformed seedlings are yellow and grow slowly.
  • the Arabidopsis thaliana seeds are planted in 1/2MS medium, and the light is 4 hours after vernalization at 4 °C for 2 hours and then at 16 h / 8 h. Culture under the cycle. Normal growth When two leaves of Arabidopsis thaliana grow, they are transplanted into a nutrient substrate and cultured.
  • ZG 3 ⁇ 43 ⁇ 4 will be transferred by T 2 of generation seeds of Arabidopsis wild type (WT) Arabidopsis seeds sterilized with ethanol and sodium hypochlorite, respectively, were seeded in OMM (control CK), 50tnM, 100mM, 200mM , 300mM NaCl concentration On 1/2 MS medium. Each concentration in the medium are seeded in different revolutions' GPJ3 ⁇ 4 due to generation of Arabidopsis and T 2 of approximately 20 seeds of wild-type Arabidopsis seeds, before sowing 4'C in the dark vernalization 2d refrigerator, then placed The culture was carried out in an incubator at 21 ° C, 16 h / 8 h photoperiod. The seed germination and growth status were observed and photographs were taken.
  • Salt stress method The concentration of NaCl aqueous solution was 400 mM, and 3 mL of brine was poured each time, once every two days, and poured three times, and observed and photographed after 15 days.
  • Drought stress method No watering, observed and photographed after 15 days.
  • the T of the transgenic Arabidopsis thaliana will be sterilized. Seeds were seeded in 1/2MS screening medium (containing Kana antibiotic at a final concentration of 50 g/mL), and cultured at 22 ° C, 16 h / 8 h photoperiod after 2 ⁇ dark vernalization for 2 days. It can be seen from Fig. 12 that the seedlings of the successfully transformed Arabidopsis thaliana grow normally, and the leaves of the untransformed seedlings are yellow and grow slowly. A total of 17 Arabidopsis thaliana are screened, and 2 of the normal growth Arabidopsis grows. When the leaves are true, they are transplanted into the nutrient substrate and continue to be cultured. T c after the generation of Arabidopsis plants grow, longer than the wild Arabidopsis plants to be strong, as shown in Fig.
  • the plants after drought stress showed that the drought-treated wild Arabidopsis plants showed dry and dead leaves after 15 days; while the growth of the transgenic lines was basically normal, and the drought tolerance of the lines 1 was higher than that of the lines 2 Strong, it is proved that the drought tolerance of transgenic Arabidopsis is significantly improved, and there is a difference in drought tolerance between strains.
  • the Ziziphusjujuba lajiaozao tissue culture seedling was provided by the Plant Cell and Embryology Laboratory of the Biotechnology Research Center of the Shanxi Academy of Agricultural Sciences.
  • Subculture medium composition MS medium + 6-BA (6-benzylaminopurine): 0.5mg/L+ agar: 6g/L, PH: 5.8-6.0 1.3 Reagents and instruments
  • the reagents used in the extraction process of saturated phenol and total R A were purchased from Shanghai Shenggong Bioengineering Co., Ltd.
  • the reverse transcription reagent kit, the fluorescent quantitative PCR kit and the PCR tubes used in the fluorescence quantification process were purchased from Dalian TaKaRa Engineering Co., Ltd.
  • the reverse transcription kit (PrimeScript® RT Master Mix) was reverse transcribed into cDNA.
  • cDNA was reverse transcribed into cDNA.
  • a relative quantitative analysis was performed using a fluorescence quantification kit (SYBR® Premix Ex TaqTM II) to calculate the relative expression A ACT value.
  • the expression amount is expressed by 2 ⁇ ( ⁇ .
  • Fluorescence quantitative PCR instrument ( ⁇ -7300)
  • each plant should select a tissue culture seedling with growth points and good growth, and then use the thoroughly disinfected scissors to cut the head of the tissue culture seedlings l-2cm away from the growth point, and quickly put the cut group seedlings into the seedlings.
  • the whole process should be operated near the alcohol lamp. If the seedlings are propagated in large quantities, it is recommended to replace all the items used every 30 minutes or burn the items on the alcohol lamp to avoid Cross-contamination.
  • all the new medium containing the seedlings should be placed in the tissue culture room so that they can grow normally, and the growth process should be observed from time to time in order to find the contaminated tissue culture in time. seedling. Usually, it is subcultured every 1 ⁇ 2 months.
  • the culture conditions are: temperature 25 ⁇ 1 °C ; photoperiod 12h/day; light intensity 2000-3000 ⁇ 1. ⁇
  • a 30-40-day-long, robust "Pepper Jujube” tissue culture seedling was immersed in a certain concentration of NaCl solution and PEG-6000 solution (simulated drought) for stress treatment, 6 plants per treatment.
  • the treatment concentration is: NaCl: 50 mM, 100 mM, 200 mM, 300 mM
  • PEG-6000 0.5MPa, 0.8MPa, 1.2MPa
  • the sampling time is -
  • NaCl 15 min, 30 min, 45 min, lh, 3 h, 7 h, 24 h, 48 h;
  • PEG-6000 15 min, 30 min, 45 min, lh, 3 h, 6 h, 24 h, 48 h.
  • the leaves and stem segments of the sample taken were separated and placed in a 10 mL centrifuge tube.
  • the liquid nitrogen was quickly frozen and stored in a -80 ° C refrigerator for total RNA extraction.
  • CTAB method was used to extract the total R A of pepper tissue cultured leaves for quantitative expression analysis.
  • RNA quality was measured by protein nucleic acid spectrophotometer to determine its purity and concentration, and formaldehyde denatured gel electrophoresis was used to test whether total RNA was degraded.
  • 1 xMOPS 500 ⁇ 600 ml Dilute 20xMOPS 20 times with DEPC treated water.
  • the preparation method of 1% agarose denaturing gel is as follows - water (treated by DEPC) 44mL
  • the volume ratio of total RNA sample to denatured Buffer was 1:3. After mixing, it was incubated in a 65 °C water bath for 10 min, placed on an ice-water mixture for 5 minutes to cool it; spotted, electrophoresed, shaken by shaking EB 40 min, SDW was washed three times, each time 15 min, UV photography.
  • the reverse transcription reaction procedure was: 37 ° C for 15 min (reverse transcription reaction), 85 ° C for 5 se C (reverse reaction of reverse transcriptase). 2.4.2 Fluorescent quantitative primer design and synthesis
  • the ZH3 gene of jujube can be used as an internal reference gene to detect mRNA expression levels of genes.
  • the Z H3 gene sequence was obtained from the sequence number EU916201, and the specific primer of the internal reference gene Zj'H3 was designed according to the obtained sequence, and a specific primer for real-time PCR was set according to the conserved region of the Z GP gene, and the gene was synthesized by Huada.
  • the primer for the internal reference gene 'H3' is:
  • the primer sequence of the target gene z GP is:
  • WP9 5 ' ⁇ C ATGGGTTGGAGATACTGGC— 3 '
  • WP10 5'- TTGGAGCAGCACTTTCACC— 3'
  • the reaction system is: lOxPCR buffer 1.5 ⁇ 1 (including Mg ion), dNTP 0.2 ⁇ 1, upstream and downstream primers each 0.5 ⁇ (20 ⁇ 1/ ⁇ 1), rTaq polymerase ⁇ . ⁇ , template cDNA ⁇ , sterilized ultrapure water plus 11.2 ⁇ 1 to The total volume is 15 ⁇ 1.
  • the reverse transcribed cDNA was diluted to 100 ng l, ⁇ was used as a fluorescent quantitative PCR template, and H3 was used as an internal reference gene.
  • the normal growth and growth of the pepper tissue culture seedlings were treated with NaCl and PEG-6000 (simulated drought), and the leaves of the jujube tissue cultured plants treated with 300 mM NaCl and 1.2 MPa PEG-6000 solution began to change after 24 h; At 48h, the leaves of the tissue cultured seedlings treated with 300 mM NaCl turned white and showed signs of death. All the leaves of the tissue cultured seedlings treated with 1.2 MPa PEG-6000 solution turned white, which died.
  • the results of spectrophotometer showed that the total RNA of the extracted material had an OD 26 o of more than 500 ng / l, 260 / 280 and The 260/230 is above 1.9, which indicates that the concentration and purity of the total RA are qualified and can be used for the next step.
  • the results of real-time PCR analysis of tissue stress after jujube seeding showed that the expression level was the highest when NaCl stress was received for 24 h, and then decreased with the prolongation of stress time, which was reduced and treated at 48 h. The amount at the beginning; the concentration increased with increasing concentration, while the 300 mM treatment was the lowest, and the morphological observations of the plants also indicated that the leaves were wilting.
  • the Zj'GP gene was up-regulated by NaCl stress for a certain period of time. When the plant was wilting, the expression decreased, so Z'GP was related to the salt tolerance of jujube seedlings.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了一种枣树谷胱甘肽过氧化物酶基因，该基因的核苷酸序列以及氨基酸序列分别如序列表中SEQ ID NO:1和SEQ ID NO:2所示。构建了包含该基因的植物表达载体，并获得转基因拟南芥植株，对其进行耐盐性和耐旱性评价。结果证实该基因作为目的基因导入植物，提高了植物的抗逆性。

Description

枣树谷胱甘肽过氧化物酶基因
技术领域
本发明涉及基因工程技术领域， 具体是一种枣树谷胱甘肽过氧化物酶基因。
背景技术
Mills最初发现动物细胞内具有抗氧化作用的 GPX, 其功能被认为是防御红血球氧化引 起的溶血， 并将其命名为谷胱甘肽过氧化物酶。 过去一直认为这是经典的 GPX, 现在把具有 这种功能的酶命名为 GPX-1.后来， Flohe研究小组纯化并分析了分布在细胞中并作用于各种 有机过氧化物及 H202的 GPX, 研究表明它是由 4个蛋白质亚基构成的四聚体， 每个亚基各 含 1个硒半胱氨酸， 并且它是以离子化硒醇 (selenol)的形式形成酶的氧化还原活性中心。大量 的研究表明， 哺乳动物的 GPXs有着较高的催化活性， 可迅速清除体内产生的多余的活性氧 (ROS ) ， 并且 GPXs 多数以谷胱甘肽 GSH(glutathione)作为底物催化分解生物体内产生的 H202。 目前， 根据 GPXs所包含的半胱氨酸残基的不同， 可简单将其分为含硒和不含硒两大 类。
植物中同样也存在属于 GPX家族的酶类， 直到最近几年， 在植物中也开展了 GPXs的 功能研究， 最初的研究是从烟草的叶片里克隆了与动物依赖硒的 GPXs 有着较高同源性的 cDNA。 后来从柑橘、 拟南芥、 和中国大白菜等多种植物中分离得到了类似基因。 研究发现， 不同于动物基因组 GPXs核苷酸 UGA终止密码子处插入的硒代半胱氨酸残基， 植物基因组 GPXs核苷酸序列携带的是一个半胱氨酸残基。动物中含硒代半胱氨酸残基的 GPXs有较强的 催化活性， 而植物中多数是半胱氨酸残基， 这就大大降低了 GPXs在植物中的催化作用。 然 而， 也有人在研究柑橘的 GPXs时， 用硒代半胱氨酸残基代替了半胱氨酸残基， 却没有出现 类似于哺乳动物的 GPXs的活性。
植物中 GPXs的结构、 对底物的特异性及在植物组织的分布上都不相同， 它们对抵御外 界胁迫起了很重要的作用， 但我们对其功能及结构特性的认识仍很有限， 还需要做大量的工 作。 因此， 克隆和鉴定植物中 GPXs, 研究抗逆机制以及与逆境相关基因的分子结构、 表达、 功能及调控， 可为充分利用这些优良抗性基因提高植物的抗逆性提供一定的实验数据和奠定 理论基础。
发明内容
本发明的目的在于提供一种枣树谷胱甘肽过氧化物酶基因， 可作为目的基因导入植物， 提高植物抗逆性， 进行植物品种改良。 本发明是采用以下技术方案实现的： 一种枣树谷胱甘肽过氧化物酶基因， 该基因的核苷 酸序列是如 SEQ ID NO: l所示的序列。
由所述的一种枣树谷胱甘肽过氧化物酶基因， 该基因的核苷酸序列编码的氨基酸序列是 如 SEQ ID NO:2所示的序列。
由所述的一种枣树谷胱甘肽过氧化物酶基因， 该基因的核苷酸序列编码的蛋白质具有如 SEQ ID NO:2所示的氨基酸序列。
所述的枣树谷胱甘肽过氧化物酶基因在提高植物抗逆性中的应用。
本发明从山西省农业生物技术研究中心园艺作物生物技术研究室构建壶瓶枣 Ziziph jujuba Mill hupingzao ) 结果枝 （即枣吊） cDNA文库中筛选到枣树谷胱甘肽过氧化物酶基因 的全序列， 将其命名为 glutathione Eero dase )。 生物信息学分析表明 基因 cDNA序列全长为 510bp， 编码 170个氨基酸， 其中包含有 16个酸性氨基酸， 19 个碱性氨基酸， 计算得知蛋白质的相对分子量为 19.26KD , 理论等电点为 5.00， 脂肪指数为 76.69。
根据枣树谷胱甘肽过氧化物酶基因 cDNA编码的序列，设计带有 EcoR I和 Sma I酶切位 点的特异性引物， 引物由上海生工生物工程技术服务有限公司合成。其上游引物为 GY1的序 列为： 5'— ACGAATTCTCATGACTAGCCAGC— 3' , 包含 限制性酶切位点 （即有下 划线部分） ， 三个保护碱基 ACG及此蛋白的起始密码子 ATG。 下游引物为 GY2的序列为： 5'— ATCCCGGGTTGAGATTCCCAAGA— 3 ' ,包含该 Sma I限制性酶切位点（即下划线部分）， 两个保护性碱基 AT及谷胱甘肽过氧化物酶的终止密码子 TGA。 经限制性内切酶 EcoR I和 Sma I修饰后连接于植物表达载体 PEZR(K)- LNY上， 然后再经含卡纳霉素的 LB平板筛选转 化子、 PCR鉴定、 酶切鉴定、 测序证明植物表达载体 PEZR K^-LNY-Z G 构建成功。 通过 冻融法将携带目的基因的植物表达载体 PEZR(K)-LNY- 转入农杆菌 LBA4404的感受态 细胞中， 经含卡纳霉素的 LB 平板筛选转化子、 PCR 鉴定、 酶切鉴定、 测序证明工程菌 PEZR Q-LNY-Z G 构建成功。农杆菌介导 基因转化拟南芥，经含卡那霉素的 1/2MS 培养基筛选获得含有目的基因 的转基因拟南芥植株 TQ代， 经 PCR鉴定目的基因证实 成功转入拟南芥。 将转 基因的丁2代拟南芥种子和野生型 （WT ) 拟南芥种子均 播种在加不同浓度 NaCl的 1 /2MS培养基上,对转 基因植株及其 T2代进行耐盐性评价， 观察其生长状况， 发现转 Z'GP 基因拟南芥植株在盐胁迫处理下长势和根系明显好于野生型 ( WT )拟南芥植株。 将长出 2片真叶的转 基因的 T2代拟南芥植株和野生型 （WT ) 拟南芥植株进行耐盐、 耐旱胁迫处理， 15天后观察其生长状况， 发现转 Z'GP 基因的拟南芥 耐盐性明显提高， 转 基因的拟南芥耐旱性明显提高。
本发明为了进一步研究 基因在枣树'体内的表达， 分别对辣椒枣组培苗进行干旱、 NaCl胁迫处理，研究逆境因素对 基因表达的影响。 CTAB法提取所辣椒枣组培苗的总 RNA, 经 OD26Q值测定、 甲醛变性胶电泳等方法检测证明所提 RNA浓度和纯度都符合标准， 质量合格。 反转录所提总 R A， 定量 PCR检测结果分析证实 可受干旱、 NaCl胁迫等 逆境因素诱导表达。
此外， 本发明为了进一步研究枣树谷胱甘肽过氧化物酶基因 Z'GP 及编码蛋白 ZjGPX, 本发明构建了重组原核表达载体 pGEX-4T-2-ZjGPX，实现其在大肠杆菌 E. coin BL21 ( DE3 ) 中的原核表达， 为该基因编码蛋白的结构、 功能、 调控等方面研究提供了一定的实验数据。
与现有技术相比， 本发明首次从枣树中分离出枣树谷胱甘肽过氧化物酶基因 ZjGPX, 该 基因作为目的基因导入植物， 提高植物抗逆性， 对于植物品种改良具有重要的现实意义， 对 于具有优良抗性的植物的抗逆机制的深入研究，有助于明确逆境相关基因的分子结构、表达、 功能及调控， 为进一步充分利用这些优良抗性基因来提高植物的抗逆性提供一定的实验数据 和奠定理论基础， 同时有助于了解 GPXs在植物活性氧信号转导中的作用， 促进人们对活性 氧作为信号分子的了解。
附图说明
图 1为 与己知 基因氨基酸同源性对比树状图。
图 2为枣树谷胱甘肽过氧化物酶的三维结构预测图。
图 3为 PCR目的基因产物图谱。
图 4 为 pGEX-4T-2及目的基因 酶切图谱。
图 5为重组质粒的酶切鉴定图。
图 6为工程菌 pGEX-4T-2-Z OP -B的 PCR鉴定图。
图 7为 pGEX-4T-2-Z GP -B 诱导产物的 SDS-PAGE分析。
图 8为 IPTG对蛋白表达量的影响与融合蛋白的纯化。
图 9为 PCR目的基因产物图谱。
图 10为 PEZR(K)-LNY及目的基因的酶切图谱。
图 11为重组质粒的酶切鉴定图谱。
图 12为筛选出的转 Z GP 基因拟南芥 TQ代幼苗。
图 13为筛选出的转 Zj'G 基因拟南芥 Tc代植株。 图 14为转 Z G 基因拟南芥 不同株系拟南芥基因检测。
图 15为转 Z GP 基因拟南芥和 WT拟南芥种子在不同浓度的 NaCl培养基上的萌发情况。 图 16为转 基因拟南芥和 WT拟南芥植株在不同浓度的 NaCl培养基上根系的生长 情况。
图 17为转 基因拟南芥和 WT拟南芥植株盐胁迫后生长情况的对比图。
图 18为转 基因拟南芥和 WT拟南芥植株干旱胁迫后生长情况的对比图。
图 19为 RNA甲醛变性胶电泳图谱。
图 20为辣椒枣组培苗经不同浓度的 NaCl胁迫后的 Z G 相对表达量的对比表。
图 21为辣椒枣组培苗经 PEG-6000胁迫（模拟千旱处理）后的 相对表达量的对比 表。
具体实施方式
实施例 1 : 枣树谷胱甘肽过氧化物酶基因 Z!GPX的生物信息学分析
本发明从山西省农业生物技术研究中心园艺作物生物技术研究室构建壶瓶枣 iziph jujuba Mill hupingzao ) 结果枝 （即枣吊、 落性枝） cDNA文库中筛选到枣树谷胱甘肽过氧化 物酶基因 的全序列。
将所测定 基因序列通过 Blastx搜索 NCBI的核苷酸数据库， 进行序列相似性分析； 用 DNAStar进行氨基酸序列比对并做此蛋白的进化树； 用 ProtPamm计算蛋白质的相对分子量 和理论等电点； 连接至 http://www.expasy.org/prosite进行保守序列分析； 将此蛋白的氨基酸序 列提交 Swiss-Model预测其三级结构。
生物信息学分析表明： 枣树谷胱甘肽过氧化物酶基因 cDNA序列为全长 510bp的开放读码 框 （ORF ) ， 通过在 NCBI数据库 Blastx搜索进行序列相似性分析表明， 该蛋白与已知的其它 植物谷胱甘肽过氧化物酶具有极高的同源性， 与蓖麻的同源性为 95%, 杨树的同源性为 93%， 拟南芥的同源性为 93%;对 Zj'GP 蛋白的氨基酸序列与其它属的物种的谷胱甘肽过氧化物酶用
DNAStar构建进化树， 如图 1所示， 枣树谷胱甘肽过氧化物酶基因的氨基酸序列和蓖麻的 GPX 亲缘关系最近， 7个物种的谷胱甘肽过氧化物酶被聚为 5个类群， 拟南芥和杨树聚为一类； 玉 米和高梁聚为一类； 枣、 葡萄、 蓖麻单独聚为一类； 聚类分析还表明枣谷胱甘肽过氧化物酶 和 6物种 GPX的氨基酸同源性均达到 73.8%以上； Zjd基因 cDNA序列全长为 510bp， 用
ProtParam分析该序列得， 它编码 170个氨基酸， 其中包含有 16个酸性氨基酸， 19个碱性氨基 酸， 计算得知蛋白质的相对分子量为 19.26KD , 理论等电点为 5.00， 脂肪指数为 76.69; .连接 至 http://www.expasy.org/prosite进行保守序列分析 , 结果表明该基因编码的蛋白具有一个谷胱 甘肽过氧化物酶活性位点， 其氨基酸序列为第 3 1个氨基酸到第 46个氨基酸 "G vLLIvNVaSkCGmT" , 还有一个谷胱甘肽过氧化物酶保守肽段， 其氨基酸序列为 "LAFPCNQF", 即第 68个氨基酸到第 75个氨基酸； 在蛋白质结构数据库中搜索到 'OP 同源 模型 2p5rA， 用 Swiss Model 程序（http：〃 au.expasy.org/tools/) 进行同源建模（homology modeling), 推测 Z G lY基因编码蛋白的三维结构如图 2所示， 枣树谷胱甘肽过氧化物酶与白杨 树谷胱甘肽过氧化物酶有极高的相似性， 推测其可能与白杨树谷胱甘肽过氧化物酶有着相似 的功能。 上述生物信息学分析方法均为本领域技术人员熟知的常规技术手段。
实施例 2: Z'G X基因原核表达载体的构建
1.材料、 试剂及仪器
1.1 载体和菌株
大肠杆菌（Escherichia coli)DH5a 菌株； 克隆载体 pSPORTl-ZjGPX、 原核表达载体 PGEX-4T-2均由山西省农业科学院生物技术研究中心园艺作物研究室保存。 _ 1.2 酶与试剂盒
DNA回收试剂盒 QIAquick®购自 QIAGEN公司； 限制性内切酶 amH I , Sma I , Sal I 、 T4DNA连接酶、 DNA Marker ( 15000bp、 2000 bp ladder)等均购自大连 TaKaRa (宝生物）工 程有限公司。
1.3 试剂及仪器设备
抗生素氨苄青霉素购自上海生工生物工程服务有限公司。 质粒提取、 琼脂糖凝胶电泳中 所用的其他试剂均购自天津天大化工。
主要的仪器设备有电子天平 （Sarorius BS200s-WEl ) 、 超净工作台 （上海迅博医疗设备 厂 Vs-840-2 )、隔水式恒温培养箱（上海市跃进医疗器械一厂 PYX-DHS )、高压灭菌锅（TOMY SS-325 ) 、 高速冷冻离心机 （Thero HP-62 ) 、 高速台式冷冻离心机 （Beck man J-25 ) 、 PCR 扩增仪（Eppendorf AG )、 UVP凝胶成像分析仪（美国 BioSpectrum 600 )、真空干燥箱（HETO VR-1 ) 、 水浴恒温振荡器 （国华企业 SHZ-82A ) 、 电热恒温水浴锅 （天津市华北实验仪器有 限公司） 、 恒温金属浴 （杭州博日科技有限公司） 、 恒温磁力搅拌器 （江苏金城国胜实验仪 器厂 79W-1 ) 、 DYY-6B型电泳仪（北京市六一仪器厂） 、 JY-DF系列电泳槽 (北京君意东方电 泳设备有限公司）、 Saritorius PH计、 分光光度计 （eppendorf BioPhotometer) 。
2. 实验方法
2.1 大肠杆菌 DH5ct感受态细胞的制作
用 CaCl2法制备大肠杆菌 （E.co/ί) DH5(感受态细胞， 具体方法见精编分子生物学实验 指南， 与细菌接触的液体及容器都需经过灭菌处理。
2.2 PCR引物的设计与合成
根据枣树谷胱甘肽过氧化物酶基因 cDNA编码的序列， 设计带有 BctrnH I和 Sma I酶切 位点的特异性引物， 引物由上海生工生物工程技术服务有限公司合成。
枣树谷胱甘肽过氧化物酶基因的上游引物 G1序列为：
5'—— TATGGATCCATGACTAGCCAGCCCA—— 3'， 包含 awH I限制性酶切位点 （即有下 划线部分）， 三个保护碱基 TAT， 及谷胱甘肽过氧化物酶 Z G 基因的起始密码子 ATG。 下 游引物 G2的序列为： 5'—— ATCCCGGGTCATGAGATTCCCAAGA—— 3'， 包含 S^m I限制 性酶切位点 （即有下划线部分）， 两个保护碱基 AT， 及谷胱甘肽过氧化物酶 基因的终 止密码子 TCA。
2.3 载体 pGEX-4T-2的制备及目的基因 Z G/^的扩增
2.3.1 碱裂解法快速提取质粒 pGEX-4T-2和 pSPORTl- Zjd
碱裂解法快速提取质粒 pGEX-4T-2和 pSPORTl -Z O ， 具体步骤见精编分子生物学实 验指南。
2.3.2 目的基因序列的扩增
以克隆载体 pSPORTl (携带目的片段） 为模板， 用设计的特异性引物进行 PCR扩增。 先 利用梯度 PCR技术设置不同的温度， 找出最佳退火温度为 61 °C。
PCR反应体系： 1 OxPCR buffer 5μ1(含 Mg2+离子）， dNTP 1 μΐ，上下游引物各 1 μ1(20ρηκ>1/ί )， rTaq聚合酶 0.3μ1， 模板 DNA Ι μΐ ( 2.3μ§/μ1 ) ， 灭菌超纯水加至总体积为 50μ1。 扩增条件为： 95 Ό预变性 5 min， 94°C变性 1 min， 61 'C退火 1 min， 72°C延伸 2 min， 共 40个循环， 最后 72 Ό 延伸 5 min。
2.4 目的基因 和载体 pGEX-4T-2的 I和 S a I酶切
利用 i^H I和^ α I分别酶切目的基因 Zj'GP 的 PCR产物和载体 pGEX-4T-2。
目的基因 的 PCR产物的酶切反应体系如下：
总体积 50μΙ
Figure imgf000008_0001
注： PCR产物的体积依据其浓度而定， 最少酶切 l〜2 g目的基因片段。 最后加超纯水至 总体积为 50μ1。
载体 pGEX-4T-2的酶切反应体系：
ΙΟχΤ buffer 2.5μ1
总体积 50μ1
Figure imgf000009_0001
分别取 5 μ1 双酶切产物，〗.0%琼脂糖凝胶电泳进行分析，确认片段大小都与预期相吻合。 2.5 酶切产物的纯化及连接
按照 DNA纯化试剂盒说明操作， 纯化双酶切后的目的基因及载体。
用 Τ4 DNA连接酶将制备好的 pGEX-4T-2载体与 ZGPJ¾因片段进行连接， 连接反应体系 总体积为 10μ1，包括 0.5 μΐ Τ4 DNA连接酶、 300 ng ZG ¾基因片段和 300 ng pGEX-4T-2， 16°C 过夜连接。
2.6连接产物的转化
将 5μ1连接产物加入到 ΙΟΟμΙ的新鲜或保存于 -80 的大肠杆菌 DH5(感受态细胞中，轻轻混 匀， 冰上放置 30min。 放入预加温到 42 'C的循环水浴中热休克 90s， 快速将管转移到冰浴中， 使细胞冷却 3-5min。 力 OO^SOC培养基， 将管转移到 37'C的摇床上， 温浴 40min使细菌复苏， 复苏期应温和的摇动细胞 （转速低于 225r/min) 。 取适当体积的转化菌液转移到含氨苄青霉 素的 LB平板， 涂匀。 将平板置于温室直至液体被吸收， 倒置平板， 37Ό静置培养过夜。
2.7 转化产物的鉴定
2.7.1 PCR鉴定方法
待转化菌在含有氨苄青霉素的 LB平板上长出菌落后， 用无菌牙签挑取转化单菌落至 50μ1 超纯水中， 沸水浴 5min使菌裂解。 取 Ιμΐ作为模板进行 PCR阳性鉴定， 反应体系为： lOxPCR buffer 1.5μ1 (含 Mg2+) ， άΝΤΡ0.2μ1, 上下游引物各 0.5μΙ (20pmol/L) ， rTaq聚合酶 Ο,ΐμΐ, 模 板 Ι)ΝΑ 1μ1 (2.3μβ/μ1) ， 灭菌超纯水加 11.2μ1至总体积为 15μ1.扩增条件同前。 将 PCR鉴定为 阳性的转化菌划线培养。 2.7.2酶切鉴定
将 PCR鉴定为阳性的转化菌用碱裂法提取质粒 DNA后，用限制性内切酶 a H I和&? / 1 对重组质粒进行酶切鉴定。
载体 pGEX-4T-2- 'G/¾ amH I和 ¾// 1双酶切反应体系：
ΙΟχΤ buffer 1.5μ1
BamH l 0.5 μΐ
Sai l 0.5μ1 总体积 ΙΟμΙ
质粒 Ι μΐ
S.D.W 7μ1
琼脂糖凝胶电泳酶切反应液， 验证酶切片段的大小， 保存携带目的片段的阳性菌株送， 并将阳性菌株往上海生工生物工程技术服务有限公司测序， 验证重组质粒读码框的正确性。 3.原核表达载体 pGEX-4T-2-ZjGPX构建的结果与分析
3.1 ZydcDNA的扩增
利用设计的特异性上下游引物进行 PCR扩增后， 产物在 1.0%琼脂糖中电泳， 经 EB染色后 在紫外灯下观测到一条约 510bp的条带， 与所设计的扩增目的片段大小吻合， 如图 3所示， 图 中： M: DL2000; 1 -2： 目的基因 PCR产物。
3.2 目的基因片段及载体片段的制备
将 PCR产物和 pGEX-4T-2质粒用 &™H I和 Sma I双酶切， 酶切产物经琼脂糖电泳， PCR 产物大小约为 510bp， pGEX-4T-2载体片段约为 4.9Kb， 与预期结果相符 （如图 4所示， 图中： Mi : DL2000 ; 1： 目的基因 OP扇切产物； M2 : DL15000 Marker; 2： pGEX-4T-2酶切产物）。 分别纯化目的片段与载体片段， 用于连接。
3.3 重组质粒的鉴定
将连接产物转入大肠杆菌感受态细胞 DH5a中后， 从含有氨苄青霉素的培养基上挑取阳 性单菌落进行 PCR鉴定， 将 PCR鉴定为阳性的克隆子用碱裂减法提取质粒 DNA后， 质粒 pGEX-4T-2-ZjGPX用 ^wzH I和 进行双酶切鉴定， 结果如图 5 所示， 图中： DL2000 Marker; 1-7 : 阳性质粒。 双酶切的片段大小约为 515bp, 与预期结果相符； 初步推断重组质 粒构建成功， 且重组质粒的大小约为 5.4Kb左右。 测序结果表明该转化菌的质粒中确实含有 目的片段， 且读码框正确， 重组质粒构建成功。
实施例 3 : Z GPAT在大肠杆菌中的表达及蛋白纯化 1 材料、 试剂及仪器
1.1 载体和菌株
大肠杆菌 (Escherichia coli)BL21(DE3)菌株； 原核表达载体 pGEX-4T-2均由山西省农业科 学院生物技术研究中心园艺作物研究室保存。
1.2 试剂盒及仪器
融合蛋白纯化试剂盒 Glutathione Resins购自大连 TaKaRa (宝生物） 工程有限公司。
本实施例用到的仪器主要有超声波仪（West Germany Elma D-7700) , DYY-6B型电泳仪 (北京市六一仪器厂） 、 DYCZ-28A型电泳槽 （北京市六一仪器厂） 、 索尼数码相机 （索尼 (中国） 有限公司 DSC-T500) 。 其他的仪器见精编分子生物学实验指南。
1.3试剂配方
融合蛋白纯化的相关试剂配方：萃取 buffer(loading buffer) :140mM NaCl; lOmM Na2HP04; 1.8mM KH2P04(pH7.5)。
洗脱 buffer: 33mM谷胱甘肽溶解在 50mM Tris-HCl (pH8.0)。 准备新鲜的。
回收 buffer:
第一种 buffer: 0.1M Tris-HCl; 0.5M NaCl(pH8.5)。
第二种 buffer: 0.1M醋酸钠； 0.5M NaCl(pH4.5)。
第三种 buffer: 140mM NaCl; lOmM lOmM Na2HP04; 1.8mM KH2P04(pH7.5)。
其他试剂及 SDS-PAGE电泳相关试剂配方见精编分子生物学实验指南。
2. 实验方法
2.1 基因工程菌 pGEX-4T-2- 'OPX-B的构建和筛选
用 CaCl2法制备大肠杆菌 BL21(DE3)感受态细胞， 具体方法见精编分子生物学实验指南。 分装成 ΙΟΟμΙ或 200μ1小份， 液氮中冷冻， -70'C贮存备用。
将实施例 2中构建的重组质粒 pGEX-4T-2-Z OPJ 和载体 pGEX-4T-2通过热激法， 导入 表达菌株 BL21 (DE3 ) 感受态细胞中， 涂于含氨苄青霉素的 LB平板， 37°C静置培养 过夜。
从 37Ό过夜培养的转化有重组质粒 pGEX-4T-2-ZjGPX的平板上分别随机挑选生长良好 的几个单菌落，进行 PCR鉴定， 方法同实施例 2，将鉴定出的阳性工程菌重新划线保存一份， 备用。
2.2 SDS-PAGE不连续系统电泳样品制备 从鉴定出的阳性工程菌 pGEX-4T-2- 'G JT-B (导入 pGEX-4T-2- 'G 质粒的 E.coli BL21 (DE3 )的菌株）与对照菌 pGEX-4T-2-B (导入 pGEX-4T-2质粒的 £.co/i BL21 (DE3 )的菌株） 进行 SDS-PAGE检测。 融合蛋白的诱导方法如下：
①分别挑取对照菌 PGEX-4T-2-B—个单菌落和阳性工程菌 pGEX-4T-2-Z^P -B几个单菌 落， 接入 10 mL含氨苄青霉素 LB液体培养基， 37'C培养过夜。
②翌日早晨按 1： 100的比例接种于相同的 LB培养基上， 37Ό培养 2h左右， 至 OD6(X) 值约为 0.5 。
③在培养物中加入终浓度为 1.0mmol/L的 IPTG, 37°C继续通气培养 4个小时。 .
④取 2 mL样品放于微量离心管中， 室温， 8000rpm离心 3 min。
⑤去上清。
⑥将沉淀重悬于 200 1 2XSDS上样缓冲液， 加 20μ1 β-巯基乙醇， 混匀。 100°C加热 5-10 min, 冰上放置约 5分钟至冷却， 取 20μ1进行 SDS-PAGE电泳。
2.3 SDS-PAGE电泳操作程序
①将玻璃板依次用自来水、 SDS、 自来水洗净， 用无水乙醇擦拭干净， 再用超纯水冲洗 干净， 晾干。 待玻璃板干后， 根据厂家的说明安装玻璃板。
②称取 1.5 g琼脂粉， 放入三角瓶， 加入 100 mL l x电泳缓冲液， 加热溶解， 置桌面上冷 却至不烫手 （约 50-60°C ) , 开始在玻璃板外边缘灌注电极胶。 电极胶装完后， 静置 15 min左 右使胶凝结。
③按照下表 1所示先配制 15 mL 15%分离胶。迅速在两玻璃板的间缝中注入分离胶溶液， 注意不要动作太大， 防止出现气泡。可用 5 mL移液器吸取胶溶液均勾灌下， 不要把胶溶液溢 到外面。 离玻璃板上端 5cm左右停止灌注， 用少许超纯水封住胶的液面将胶面压平， 将凝胶 垂自放置于室温约 30 min待胶完全凝固。
表 1 SDS-PAGE分离胶和浓缩胶成分表
Figure imgf000012_0001
1.5 Tris-HCI (pH8.8) 3.8 mL ( 1.5M)
l.OMTris-HCI (pH6.8) 0.63 mL
TEMED 0.006 mL 0.005 mL
④用滤纸吸干液面上的水， 按照表 2.1所示配制 5 mL 5%的浓缩胶， 灌注至离玻璃板上端 2 mm左右停止灌注， 立即在浓缩胶溶液中插入干净的梳子， 避免混入气泡， 将凝胶垂直置室温 下。
⑤待胶凝结完全后，小心拔出梳子，立即用去离子水洗涤加样槽以去除未聚合的沉凝物， 一小时左右后， 在电泳槽中加入 l x电泳缓冲液。
⑥用微量移液器加样分别加样 20μ1至加样槽。
⑦打开电泳仪， 开始电泳， 浓缩胶稳压 70 ν， 但是电流不要超过 20 mA, 当凝胶中溴酚 蓝到达分离胶时升压至 150 V继续电泳， 当溴酚蓝电泳至凝胶底部 （即分离胶底部） 1 cm处， 电泳结束。
⑧将胶卸下， 左下切角作为正反面标记， 加入适量的固定液进行固定处理。
⑨凝胶固定约 1.5 h， 考马斯亮蓝 R-250染色 2 h, 最后脱色至背景清晰为止。
⑩照相保存， 观察分析结果。
2.4 GST-Zj'Gi Y融合蛋白表达诱导条件的优化
2.4.1 IPTG诱导时间对 GST-Z GP 融合蛋白表达的影响
选取鉴定出的阳性工程菌 pGEX-4T-2-Z G -B， 37°C振荡培养至 OD6(W值为 0.5左右， 力口人 IPTG至终浓度为 1.0 mmol/L， 37°C诱导培养， 于 1 h、 2 h、 3 h、 4 h、 5 h、 6 h取出 2 mL 菌液， 以不含插入子的 pGEX-4T-2-B作为空白对照， 对照菌 pGEX-4T-2-B同样加人 IPTG至 终浓度为 l.O mmol/L, 37'C诱导培养， 于 4 h取出 2 mL菌液， 按上述样品制备的方法制备样 品， 每个样品取 20 μ1， 进行 SDS-PAGE检测， 分析 IPTG在不同的诱导时间下对蛋白表达量 的影响。
2.4.2 IPTG诱导浓度对 GST-Zj' 融合蛋白表达的影响
选取鉴定出的阳性工程菌 pGEX-4T-2-Z OP -B， 37°C振荡培养至 OD6TO值为 0.5左右， 加入 IPTG至终浓度分别为 0.1、 0.4、 0.8、 1.0、 1.2 、 1.6mmol/L, 37°C诱导培养 4 h， 以不 含插入子的 pGEX-4T-2-B作为空白对照， 对照菌 PGEX-4T-2-B加入 IPTG至终浓度为 1.0 mmol/L, 按上述样品制备的方法制备样品， 每个样品取 20 μ1， 进行 SDS-PAGE检测， 分析 IPTG在不同诱导浓度下对蛋白表达量的影响。
2.5 融合蛋白 GST- 'Gi¾的纯化
细胞抽提物的制备、 GST-ZG 融合蛋白的纯化及 GST纯化柱的再生，具体操作程序见 融合蛋白纯化试剂盒 Glutathione Resins说明书。
3. ZG 蛋白原核表达及纯化结果与分析
3.1 工程菌 pGEX-4T-2-ZGP -B的筛选
将构建成功的原核表达载体 pGEX-4T-2-Z/Gi Y转入大肠杆菌 BL21 (DE3)后， 从筛 选平板上挑取单菌落经 PCR鉴定， 琼脂糖凝胶电泳结果表明， 在 510bp处有明显的目的基因扩 增条带， 与预期结果一致， 如图 6所示， 图中 M: DL2000Marker; 1-6： 阳性菌落。 证明带有 目的基因 的载体 pGEX-4T-2- 'GP 成功转入大肠杆菌 (E.coli) BL21 (DE3) 中。 3.2工程菌 pGEX-4T-2-Z'Gi y-B表达融合蛋白的 SDS-PAGE分析
工程菌 pGEX-4T-2-ZOP B经 IPTG诱导 4 h后取样， 电泳结果如图 7所示， 图中 M: 蛋白 Marker; 13： 对照空菌体； 5、 6、 9、 10： 表达有 45KD融合蛋白的阳性菌体。 经 SDS-PAGE 电泳观察到含有质粒 pGEX-4T-2-Zj'GP 的大肠杆菌 BL21 (DE3)在 IPTG诱导的条件下， 与对 照空菌体 PGEX-4T-2-B相比在大约 45KD处产生一条特异性蛋白条带， 表明诱导枣树谷胱甘肽 过氧化物酶表达成功。 （GST大小约为 26KD, 目的蛋白大小为 19.26KD, 故融合蛋白大小约为 45KD)。
3.3 IPTG不同诱导时间和浓度对蛋白表达量的影响及融合蛋白纯化结果
OD600值约为 0.5的工程菌 pGEX-4T-2-Z/GP -B新鲜菌液中加人 IPTG至终浓度为 1.0 mmol/L, 37°C诱导培养， 分别于 1 h、 2h、 3 h、 4h、 5 h、 6 h取出2 mL菌液。
OD 值约为 0.5的工程菌 pGEX- 4T-2- 'GP -B新鲜菌液中加入 IPTG至终浓度分别为 0.1、 0.4、 0.8、 1.0、 1.2 、 1.6mmol/L, 37Ό诱导培养 4h， 取菌液。
将采取的菌液处理并进行 SDS-PAGE电泳。 电泳结果如图 8所示， IPTG诱导时间为 5h时， 目的蛋白表达量最大； IPTG终浓度为 0.4 mmol/L时， 目的蛋白表达量最大。 然后大量表达融 合蛋白， 收集细菌裂解液， 利用亲和层析技术， 与 GST标签蛋白纯化树脂结合， 经多次洗脱 纯化， 获得分子量为 45KD的 GST-ZGP 融合蛋白， 第一条带， 可见 GST融合蛋白与 GST标签 蛋白纯化树脂有很高的亲和效率。 见图 8, 图中 M:蛋白 Marker; 1： 纯化的 GST-ZG/ Y融合蛋 白； 2:对照 GST蛋白表达； 3-8:不同时间诱导 GST-Zj'G 融合蛋白表达， 诱导时间分别是 lh、 2h、 3h、 4h、 5h、 6h; 9-14： 不同浓度诱导 GST-ZOP 融合蛋白表达， IPTG至终浓度分别 为 0.1、 0.4、 0.8、 1.0、 1.2 、 1.6mmol/Lc
实施例 4: 2'6 ¾因植物表达载体的构建
1. 实验材料
1.1 载体及菌株
植物表达载体 PEZR(K)-LNY (含黄色荧光蛋白（YFP)标记基因）、大肠杆菌 ( co//)DH5a 菌株、 含 Z'G¾f基因的重组质粒 pSPORTl-ZjGPX均由山西省农业科学院生物技术硏究中心 园艺作物研究室保存。
1.2 酶与试剂盒
DNA回收试剂盒 TaKaRa DNA Fragment Purification Kit Ver.2.0, 限制性内切酶 " H I、 Smal、 EcoR I ^ HindllL T4DNA连接酶 (DNA Ligation Kit Ver.2.0) 、 DNA Marker等均购 自大连 TaKaRa (宝生物） 工程有限公司。
1.3常用试剂及培养基
1、 卡那青霉素 （100mg/mL)
2、 酚：氯仿：异戊醇 （ V： V： V， 25： 24： 1 )
3、 葡萄糖溶液 （1 M)
4、 EDTA (0.5M, pH8.0 )
5、 ΙΟχΤΒΕ电泳缓冲液： 硼酸 55.2g/L
Tris 108g/L
EDTA(0.5M, pH8.0) 40mL
6、 10% SDS (pH7.2)
7、 NaOH (2M)
8、 Tris-HCl (1 M， pH8.0)
9、 醋酸钠 （3M， pH5.2)
10、 TE溶液 （lOmMTris-Hcl, ImM EDTA, pH8.0)
11、 LB培养基： （固体培养基加琼脂 0.15g/L)
蛋白胨 0.1g/L
酵母提取物 0.05g/L
NaCl 0.05g/L
2. 实验方法 2.1 大肠杆菌 CE.cO/0DH5a菌株感受态细胞的制作
用 CaC 制备大肠杆菌 (£.CO/0DH5(x菌株感受态细胞， 具体方法见精编分子生物学实验 指南。
2.2 PCR引物的设计与合成
根据枣树谷胱甘肽过氧化物酶基因 cDNA编码的序列，设计带有 EcoR I和 Sma I酶切位 点的特异性引物， 引物由上海生工生物工程技术服务有限公司合成。
上游引物为 GY1 : 5'— ACGAATTCTCATGACTAGCCAGC— 3' , 包含 £co H限制性酶 切位点 （即有下划线部分） ， 三个保护碱基 ACG及此蛋白的起始密码子 ATG。
下游引物为 GY2: 5'— ATCCCGGGTTGAGATTCCCAAGA— 3' , 包含该 Sma I限制性酶 切位点 （即下划线部分） ， 两个保护性碱基 AT及谷胱甘肽过氧化物酶的终止密码子 TGA。 2.3 载体 PEZR(K)-LNY和 pSPORTl- ZjG 的制备及目的基因 ZjGPX的扩增
2.3.1 质粒 PEZR(K)-LNY和 pSPORTl— 的制备
pSPORTl-Z G/ ¾r质粒的 DH5a菌液用 2mL的 LB液体培养基加 Ιμΐ氨苄青霉素 （100 mg/mL )接菌后 37°C过夜培养所得。 含 PEZR(K)-LNY质粒的 DH5a菌液则用 2mL的 LB液 体培养基加 Ι μΐ卡那青霉素 （100 mg/mL ) 接菌后 37Ό过夜培养所得。
碱裂解法快速提取质粒 PEZR(K)-LNY和 pSPORTl-Z GP 的具体步骤见精编分子生物学 实验指南。
最后将干燥的质粒溶于 20 μΐ TE中， -20°C保存备用。
2.3.2 目的基因 的扩增
以克隆载体 pSPORTl (携带目的片段） 为模板， 用设计的特异性引物进行 PCR扩增。 先 利用梯度 PCR技术设置不同的温度， 找出最佳退火温度为 52Ό。
PCR反应体系： 1 OxPCR buffer 5μ1 (含 Mg2+离子）， dNTP 1 μΐ，上下游引物各 1 μΚ 20pmol/L )， rTaq聚合酶 0.3μ1， 模板 DNA Ι μΐ (2.3μ§/μ1) ， 灭菌超纯水 40.7μ1， 总体积 50μ1。 扩增条件为： 95 °C预变性 5 min， 94°C变性 1 min， 52'C退火 1 min， 72°C延伸 2 min， 共 40个循环， 最后 72°C 延伸 5 min。
2.4 目的基因 G IY和载体 PEZR(K)-LNY的 £coi? I和 Swa I酶切
目的基因 的 PCR产物两端及载体 PEZR(K)-LN Y的多克隆位点都具有 EcoR I和 Sma I限制性内切酶点， 利用 EcoR I和 I分别酶切目的基因 Zj'G /^的 PCR产物和载体 PEZR(K)-LNY, 使它们具有相同的粘性末端， 为连接做好准备。 目的基因 Z G ¾PCR产物的酶切反应体系如下:
50μ1
Figure imgf000017_0001
注： PCR产物的体积依据其浓度而定， 最少酶切 1〜2μ§目的基因片段。 最后加超纯水至 总体积为 50μ1。
载体 PEZR(K)-LNY的酶切反应体系如下：
总体积 50μ1
Figure imgf000017_0002
酶切后分别取 5 μΐ双酶切产物， 1.0%琼脂糖凝胶电泳进行分析， 确认片段大小都与预期 相吻合。
2.5 酶切产物的纯化
按照 DNA纯化试剂盒说明操作， 纯化双酶切后的目的基因及载体。
目的基因 片段和载体 PEZR(K)- LNY经 EcoR I和 Sma I双酶切后，按照琼脂糖凝 胶 DNA回收试剂盒 TaKaRa DNA Fragment Purification Kit Ver.2.0的说明书进行纯化操作。
2.6 酶切产物的连接
用 T4 DNA连接酶将纯化后的 PEZR(K LNY载体与 因片段进行连接。将质粒载体 PEZR(K)-LNY的 DNA片段与目的基因 Z'OP 的 DNA片段混合制备成体积为 5μ1左右的 DNA溶 液 （载体 DNA和插入 DNA的摩尔数比一般为： 0.03pmol : 0.1〜0.3pmol ) , 向上述 DNA溶液 中加入等体积的 DNA Ligation Kit中的 Solution I， 充分混匀。 连接反应体系总体积为 10 μΐ , 充分混匀后， 16°C反应过夜。
2.7连接产物的转化 将 ΙΟΟμΙ的新鲜或保存于 -70Γ的大肠杆菌感受态细胞 DH5a加入到上面的连接产物中， 轻 轻混匀， 冰上放置 30min。放入预加温到 42°C的循环水浴中热休克 90s, 快速将 EP管转移到冰 浴中， 使细胞冷却 3〜5min。 力 B400 μΐ的 LB培养基， 将 EP管转移到 37°C的摇床上， 温浴 40 min 使细菌复苏， 复苏期应温和的摇动细胞（转速低于 225转 /min) 。 将适当体积的已转化感受态 细胞转移到 LB平板 （含氨苄青霉素） ， 涂匀。 将平板置于室温直至液体被吸收， 倒置平板， 37。C静置培养过夜。
2.8 PCR鉴定
待转化菌在含有卡那霉素抗性的 LB平板上长出军落后，用无菌牙签挑取转化单菌落至 50μ1 超纯水中， 沸水浴 5min使菌裂解。 取 Ιμΐ作为模板进行 PCR阳性鉴定， 反应体系为： lOxPCR buffer 1.5μ1 (含 Mg2+) ， άΝΤΡ0.2μ1, 上下游引物各 0.5μ1 (20pmol/L) ， rTaq聚合酶 Ο.ΐμΐ, 模 板 DNA Ιμΐ (2.3μ^μ1) ， 灭菌超纯水加 11.2μ1至总体积为 15μ1.扩增条件同前。 将 PCR鉴定为 阳性的转化菌划线培养。
2.9重组表达载体的酶切鉴定
将 PCR鉴定为阳性的转化菌用碱裂法提取质粒 DNA， 用限制性内切酶对重组质粒进行 HwHII和 Sa HI酶切鉴定， 电泳观察结果， 并将阳性菌株送往往华大基因公司测序， 验证 重组质粒读码框的正确性。
Hindli BamHl的酶切体系如下：
ΙΟχΚ buffer Ιμΐ
HindWl 0.5μ1
BamHl 0.5μ1 总体积 ΙΟμΙ
重组质粒 Ιμΐ
S.D.W 7μ1
3. Z'GAY基因植物表达载体构建的结果与分析
3.1 Z'GP cDNA的扩增
利用设计的特异性上下游引物进行 PCR扩增后， 产物在 1.0%琼脂糖中电泳， 经 EB染色后 在紫外灯下观测到一条约 510bp的条带， 如图 9所示， 图中， Ml: DL15000Marker; 1： PCR 目的基因酶切产物， 与所设计的扩增目的片段大小相符， 说明已成功克隆到 ZGPJ¾因。 3.2 目的基因片段和载体片段的制备
用 I和>¾^ I双酶切 G¾因的 PCR产物和 PEZR(K)-LNY表达载体， 酶切产物进 行琼脂糖凝胶电泳， 结果如图 10所示， 图中， M: DL15000 arker; 1： 目的基因酶切产物； 2： PEZR(K)-LNY酶切产物， PCR产物大小约为 510bp， PEZR(K)-LNY载体片段分别约为 11.7Kb, 与预期结果相符。 分别按试剂盒说明纯化目的片段与载体片段， 用于连接。
3.3 重组质粒的鉴定
将连接产物转化大肠杆菌 DH5a，从含有卡那霉素的培养基上挑取阳性单菌落进行 PCR鉴 定。提取阳性克隆的质粒 DNA,用 H III和 awH I双酶切，产生 1个条带，其大小约为 11.7Kb (如图 11所示， 图中， M: DL15000 Marker; 1-10: 阳性质粒)， 表明载体中已经带有 因的目的片断。 另外， 测序结果也表明， 重组质粒的外源插入片段的序列正确。.上述结果证 实， Z GP 基因已经成功构建到植物表达载体 PEZR(K)-LNY上， 并将该载体命名为
PEZR(K)-LNY-Zj'GP 。
实施例 5: 农杆菌介导 基因转化拟南芥
1.材料、 试剂与仪器
1.1 植物材料
野生型拟南芥种子来源于山西省农业科学院生物技术研究中心园艺作物研究室， 培养基 质购自山西省农科园艺作物研究所。
1.2载体和菌株
植物表达载体 PEZR(K)-LNY- d、根癌农杆菌 LBA4404均由山西省农业生物技术研究 中心园艺作物研究室保存。
1.3 试剂及仪器设备
卡纳霉素购自上海生工生物工程服务有限公司； ΥΕΒ、 1/2 MS培养基中所用试剂均购自 天津天大化工。
人工气候培养箱（SANYO,MLR-350)、索尼数码相机（索尼（中国）有限公司 DSC-T500)。
1.4培养基及试剂配方
1.4.1 YEB培养基：
蛋白胨 0.05g/L， 酵母膏 0.01 g/L, 蔗糖 0.05g/L， 硫酸镁 0.005g/L。
注- 固体培养基需加终浓度为 1.0°/。的琼脂粉。
1.4.2 1/2MS培养基：
按配制 MS培养基配制 1/2MS培养基， 大量元素、微量元素、有机元素、铁盐的量各减半， 蔗糖 30g/L， 琼脂粉 6g/L。 2.实验方法
2.1 拟南芥的培养
将拟南芥种子先在 95%乙醇浸泡 30〜60s; 再转入 2.65%次氯酸钠灭菌 5min， 其间上下颠 倒洗， 5000rpm离心 2min; 灭菌水洗三次； 然后将种子悬浮于 0.1%的琼脂粉溶胶中； 用枪头 吸上悬浮液将种子点播于 1/2MS固体培养基上， 封口； 4°C黑暗春化 2d后， 将拟南芥种子在 2TC , 16 h / 8 h光周期条件下进行培养； 当拟南芥长出 2片真叶时， 移栽到营养基质中继续培 养。
2.2 根癌农杆菌 LBA4404感受态细胞的制作
制备根癌农杆菌 LBA4404感受态细胞的具体步骤见精编分子生物学实验指南。
2.3 工程菌 PEZR(K)-LNY-Z OP -L的构建及筛选
通过冻融法将构建成功的重组质粒 PEZR(K)-LNY-Z G^¥转入农杆菌 LBA4404, 提取阳性 克隆的质粒 DNA， 并通过 PCR检测重组质粒是否转入农杆菌。
从 28'C培养的转化有重组质粒 PEZR(K)-LNY-Z G ^的筛选平板上随机挑选生长良好的 若干单菌落， 各取二分之一菌落至 50μ1超纯水中， 沸水浴 5min使菌裂解， 取 Ι μΐ作为模板 进行 PCR鉴定。 将鉴定出的阳性工程菌重新划线保存一份， 备用。
2.4工程菌 PEZRCIQ-LNY-Z GP -L的培养
挑选鉴定正确的阳性克隆于 lOmLYEB液体培养基 (含终浓度为 50 g/mL的卡纳抗生素)过 夜培养后， 6000 rpm离心 5min收集菌体， 用培养基悬浮菌体至 OD6<M)值为 0.8左右， 用于转化拟 南芥植株。
2.5 农杆菌侵染拟南芥
拟南芥植株形成花蕾时， 即可被用于农杆菌转化。将含有农杆菌的转化介质倒入烧杯中， 将拟南芥植株的花蕾部分浸入转化介质 3〜5S， 浸染过的植株放到塑料盘中， 用薄膜覆盖， 暗 处放置 12h。然后揭开薄膜，正常条件下继续培养转化过的拟南芥植株。当拟南芥的角果枯黄， 欲开裂时， 收获种子， 此为 TQ代种子。
2.6 转 基因拟南芥的筛选
将消毒的转基因拟南芥植株 Tc代种子播种于 1/2MS培养基 (含终浓度为 50 g/mL的卡纳 霉素 4°C黑暗春化 2d后置于 22'C,16h/8h光周期下培养。 转化成功的拟南芥种子长出来的 幼苗生长正常， 未转化成功的幼苗叶子发黄， 生长缓慢。 当生长正常的拟南芥长出 2片真叶 时， 移栽到培养基质中继续培养至角果枯黄、 欲裂开， 收取 ^代种子。 将 ^代种子的拟南 芥播种于 1/2MS培养基 ,' 4°C黑暗春化 2d后在 16 h / 8 h光周期下培养。 生长正常的 拟南芥长出 2片真叶时， 移栽到营养基质中继续培养。
2.6 转 基因拟南芥的胁迫处理
2.6.1 转 基因拟南芥种子的耐盐性实验
将转 Z G ¾¾因拟南芥的 T2代种子和野生型 （WT) 拟南芥种子用乙醇和次氯酸钠灭菌， 均分别播种在 OmM (对照 CK)、 50tnM、 100mM、 200mM、 300mM浓度 NaCl的 1/2MS培养基 上。 每一个浓度的培养基分别播种不同转 'GPJ¾因拟南芥的 T2代和野生型拟南芥种子约 20 颗种子， 播种后先在 4'C冰箱内黑暗春化 2d, 然后再放入 21 °C， 16 h / 8 h光周期条件的培养箱 内进行培养。 观察其种子萌发和生长状况并记录拍照。
2.6.2 转 基因拟南芥植株的耐盐、 耐旱性实验
将转 Ζ ^ΛΥ基因拟南芥的 Τ2代种子和野生型 （WT) 拟南芥种子用乙醇和次氯酸钠灭菌， 分别播种 1/2MS培养基上。 播种后先在 4Ό冰箱内黑暗春化 2d， 然后再放入 21 Ό， 16 h /. 8 h光 周期条件的培养箱内进行培养。当拟南芥植株长出 2片真叶时， 移栽到营养基质中继续在相同 条件下培养， 移入基质 3周后幵始进行耐盐、 耐旱胁迫处理。
盐胁迫方法： NaCl水溶液浓度为 400mM, 每次每棵浇 3mL的盐水， 隔两天浇一次， 共 浇了三次， 15天后观察并拍照。
干旱胁迫方法： 不浇水， 15天后观察并拍照。
3. 实验结果
3.1 转基因拟南芥植株的获得
按 2.5的方法侵染拟南芥后， 将消毒的转基因拟南芥的 T。代种子播种于 1/2MS筛选培养基 (含终浓度为 50 g/mL的卡那抗生素） ， 4Ό黑暗春化 2d后在 22'C， 16h / 8h光周期下培养。 从图 12可知， 转化成功的拟南芥种子长出来的幼苗生长正常， 未转化成功的幼苗叶子发黄， 生长缓慢， 共筛选出 17株拟南芥， 生长正常的拟南芥长出 2片真叶时， 移栽到营养基质中继续 培养。 长大后的 Tc代拟南芥植株， 长的比野生拟南芥植株要壮， 如图 13所示。
将消毒的转基因拟南芥植株的 代种子播种于 1/2 MS培养基， 4°C黑暗春化 2d后在 2TC , 16 h / 8 h光周期下培养。 生长正常的拟南芥长出 2片真叶时， 移栽到营养基质中继续 培养。 结果如图 14所示， 图中 M: DL5000Marker; 1-10： 代拟南芥不同株系。 10个株系 都均为转 基因株系。
3.3 转 ' /¾因拟南芥的抗性鉴定
3.3.1 转 GPJ¾因拟南芥种子的耐盐性鉴定 观察种子的萌发情况发现， 播种后 9天， 50mM， lOOmM NaCl的培养基上种子萌发并生长 出绿色叶片； 而 200mM， 300mM NaCl培养基上种子仅有萌出白色小芽， 均没有发出绿色叶 片发芽， 如图 15所示。 播种 15天后观察植株的生长情况， 发现部分转基因拟南芥的幼苗生长 势和根系明显好于野生拟南芥， 但是 200mM， 300mM NaCl处理依然没有种苗生长， 如图 16 所示。
3.3.2转 ZjGPX基因拟南芥种子的耐盐、 耐旱性鉴定
如图 17所示： NaCl胁迫 15天后的植株， 可见野生型拟南芥植株叶片己失水萎焉， 有了 枯萎的迹象：而转 Zj'OP 株系 1和株系 2植株的叶片和植株仍然表现正常。证明转 Zj'OP 基 因的拟南芥耐盐性明显提高。
如图 18所示干旱胁迫后的植株， 15天后发现干旱处理的野生拟南芥植株表现出叶片干 枯、 死亡； 而转基因株系的植株生长基本正常， 株系 1的耐旱性比株系 2强， 证明转 基因的拟南芥耐旱性明显提高， 并且株系间的耐旱性存在差异。
实施例 6: ZjGPX在枣树体内的表达
1.材料、 试剂及仪器 1
1.1植物材料
辣椒枣 (Ziziphusjujuba lajiaozao ) 组培苗由山西省农业科学院生物技术研究中心植 物细胞与胚胎研究室提供。
1.2培养基及主要试剂配方
继代培养基成分： MS培养基 + 6-BA ( 6-苄氨基嘌呤）：0.5mg/L+琼脂: 6g/L， PH:5.8-6.0 1.3 试剂及仪器
饱和酚、 总 R A提取过程中所用到的试剂均购自上海生工生物工程有限公司。 反转录试 剂盒， 荧光定量 PCR试剂盒及荧光定量过程中所用的 PCR管均购自大连 TaKaRa (宝生物） 工 程有限公司。
反转录试剂盒 (PrimeScript® RT Master Mix)反转录成 cDNA; 以 cDNA为模板，利用荧 光定量试剂盒（SYBR® Premix Ex Taq™ II )进行相对定量分析，计算相对表达量 A ACT值， 以 2· (ΔΔ 表示表达量。 荧光定量 PCR仪 (ΑΒΙ-7300 )
2. 实验方法
2.1 样品的处理 组培苗繁殖的整个过程均在超净台进行， 每次操作前都需将所用的物品和器皿进行彻底 消毒或在酒精灯上灼烧。
继代培养时每一株均要选取有生长点且长势好的组培苗， 然后用彻底消毒的剪刀截取距 生长点 l-2cm的组培苗头部，并迅速将剪下的小组培苗放入己灭菌的继代培养基中，整个过程 要在酒精灯附近操作， 若大批量繁殖组培苗， 建议每 30分钟更换所用的所有物品或将所用物 品在酒精灯上灼烧， 从而避免交叉污染。
若所有组培苗均已更换培养基， 则要将全部含有小组培苗的新培养基放入组织培养室， 使其可以正常生长， 生长过程要不定期进行观察， 以便及时发现污染的组培苗。 通常情况每 1~2个月继代培养一次。
培养条件为： 温度 25±1 °C ; 光照周期 12h /天； 光照强度 2000-3000μιηο1.ιη
2.2材料的准备及采集
选取 30-40 天长势较一致、 健壮的 "辣椒枣"组织培养苗浸入一定浓度的 NaCl 溶液和 PEG-6000溶液 （模拟干旱） 中胁迫处理， 每个处理 6株。
处理浓度为： NaCl: 50mM、 100mM、 200mM、 300mM
PEG-6000: 0.5MPa、 0.8MPa、 1.2MPa
然后分别在不同的时间取样， 取样时间为-
NaCl: 15min、 30min、 45min、 lh、 3h、 7h、 24h、 48h;
PEG-6000: 15min, 30min、 45min、 lh、 3h、 6h、 24h、 48h。
将所取样品的叶和茎段分开， 分别放入 10mL离心管中， 液氮速冻后保存于 -80'C冰箱， 用于总 RNA的提取。
2.3 植物总 RNA的抽提和质量检测
2.3.1 植物材料 R A的提取
CTAB法提取辣椒组培苗叶片的总 R A， 用于定量表达分析。
2.3.2 R A质量检测
总 RNA质量检测用蛋白核酸分光光度计检测其纯度和浓度，同时用甲醛变性胶电泳法来 检验总 RNA是否降解。
( 1 ) 甲醛变性胶电泳
RNA甲醛变性电泳液：
1 xMOPS 500〜600 ml: 用 DEPC处理过的水稀释 20xMOPS 20倍。 1 %琼脂糖变性胶配制方法如下- 水 （经 DEPC处理） 44mL
甲醛 3.0mL
20xMOPS 3.0mL
琼脂糖 0.6g
加热溶解后再加水至总体积为 60ml， 使溶液冷却。
注： 制胶时先将水、 MOPS、 琼脂糖融化， 室温放置至冷却到 60°C， 再加入甲醛， 混匀 并置通风橱中 15分钟后倒入制胶器中。
(2) 总 RNA变性 Buffer制备方法：
20xMOPS 5μ1
去离子甲酰胺 50μΙ
甲醛 16μ1 总体积 87μ1
甘油色素 16μ1
Figure imgf000024_0001
总 RNA样品与变性 Buffer的体积比为 1 : 3， 混匀后在 65°C水浴中温浴 10min， 置于冰水混 合物上 5分钟使之冷却； 点样， 电泳， 经摇床慢摇 EB染色 40min， S.D.W洗三次， 每次 15min， 紫外照相检测。
2.4 R A的反转录及荧光定量 PCR
2.4.1 R A的反转录
按试剂盒 PrimeScript® RT Master Mix说明将检测合格的总 R A进行反转录，反转录体系 如下-
5 X Prime Script Buffer 4μ1
Total RNA
S.D.W up to 20μ1
反转录的反应程序为： 37°C 15min (反转录反应）， 85°C 5seC (反转录酶的失活反应）。 2.4.2荧光定量引物设计与合成
本研究室孙海峰等研究表明枣树 ZH3基因可作为内参基因对 基因进行 mRNA表 达水平的检测。 由序列号 EU916201获得 Z H3基因序列， 根据所得序列设计内参基因 Zj'H3 的特异性引物， 根据 Z GP 基因保守区设置用于荧光定量 PCR的特异性引物， 并委托华大 基因合成。 内参基因 'H3的引物为：
Hi： 5'— GAGGAAGCAACTGGCAACTAAGG— 3'；
¾： 5 '—ACC AGCCTCTGGAATGGAAGTTTG— 3 '。
目的基因 z GP 的引物序列为：
WP9: 5 '― C ATGGGTTGGAGATACTGGC— 3 '
WP10: 5'— TTGGAGCAGCACTTTCACC— 3'
2.4.3荧光定量 PCR分析
以反转录产物 cDNA为模板， 分别以 WP9和 WP10; 和¾为引物， 用荧光定量 PCR 仪检测各种胁迫处理后枣树谷胱甘肽过氧化物酶基因的表达情况。反应体系为： lOxPCR buffer 1.5μ1 (含 Mg离子) , dNTP 0.2μ1, 上下游引物各 0.5μΓ(20ριηο1/μ1) ， rTaq聚合酶 Ο.ΐμΐ, 模板 cDNA Ιμΐ, 灭菌超纯水加 11.2μ1至总体积为 15μ1。
2.4.4 实时荧光定量
将反转录的 cDNA稀释为 100ng l， 取 Ιμΐ作为荧光定量 PCR模板， 用 H3作为内参基因， 实时定量 RT-PCR反应所用试剂盒为 SYBR Green l (TaKaRa)。在实时荧光定量 PCR仪上进行 相对定量分析。 20μ1反应体系中加入：上下游各引物 0.4μ1 (20ρηιο1/μ1)， cDNA 1 μ1， rTaq ΙΟμΙ, Rox 0.4μ1, 用水加至 20μ1; 每一个样品均做 3个重复， PCR的反应程序设置参照荧光定量试剂 盒说明书。 反应结束后， 整理实验结果， 按照公式 Χ=2·ΔΔα进行目的基因的相对定量。
3.实验结果
3.1 组培苗胁迫处理后形态学观察结果
将正常生长且长势较一致的辣椒组培苗进行 NaCl和 PEG-6000 (模拟干旱）处理， 处理至 24h时 300mM NaCl和 1.2MPa PEG-6000溶液处理过的枣组培苗叶片开始变蔫； 处理至 48h时， 300mM NaCl处理的枣组培苗叶片变白， 有死亡迹象， 1.2MPa PEG-6000溶液处理的组培苗叶 片全部变白， 即死亡。
3.2 总 RNA的质量检测结果
3.2.1 R A的 OD值
' 将所提取到的总 R A按 1 : 49与 RNA-free water配好后， 用分光光度计检测结果表明， 所 提材料的总 RNA的 OD26o均在 500ng/ l以上， 260/280和 260/230均在 1.9以上， 表明所提总 R A 的浓度和纯度都合格符合要求， 可以用于进行下一步检测。
3.2.2 甲醛变性胶电泳 将所提 RNA样品处理后， 进行甲醛变性胶电泳，凝胶成像分析仪下观察电泳结果， 如图 19所示， 可以看到较分明的两条带， 且前后两条带亮度比接近 2:1， 表明所提 RNA可用于反 转录。
3.3 定量 PCR检测结果：
如图 20所示， NaCl胁迫枣组培苗后的荧光定量 PCR分析结果， 表明 在收到 NaCl 胁迫 24h时表达量最高， 而后随着胁迫时间的延长表达量下降， 48h时已降至与处理开始时 的量； 不同浓度之间随着浓度的升高表大量增加， 而 300mM处理表达量最低， 这时植株的 形态学观察结果也表明叶片萎蔫。 说明 Zj'GP 基因受 NaCl胁迫一定时间后上调， 当植株萎 蔫时表达量下降， 因此 Z'GP 与枣苗的耐盐性有关。
如图 21所示， PEG-6000胁迫辣椒枣组培苗后的荧光定量 PCR分析结果。 在受到 PEG-6000胁迫时， 处理初期 30-45min降至最低， lh幵始上升， 7h时表达最高， 而后随着胁 迫时间的延长表达量下降， 48h时 0.8MPa和 1.2MPa处理已经没有表达， 0.5MPa处理表达量 也几乎降为零， 此时植株已经变白有死亡迹象。 说明 基因受模拟干旱胁迫后上调与枣 苗的耐旱性有关， 当植株受干旱致死时表达下降至零。

Claims

权 利 要 求
1.一种枣树谷胱甘肽过氧化物酶基因， 其特征是该基因的核苷酸序列是如 SEQ ID ΝΟ:1 所示的序列。
2.根据权利要求 1 所述的一种枣树谷胱甘肽过氧化物酶基因， 其特征是该基因的核苷酸 序列编码的氨基酸序列是如 SEQ ID NO:2所示的序列。
3.根据权利要求 1 所述的一种枣树谷胱甘肽过氧化物酶基因， 其特征是该基因的核苷酸 序列编码的蛋白质具有如 SEQ ID NO:2所示的氨基酸序列。
4. 根据权利要求 1所述的一种枣树谷胱甘肽过氧化物酶基因， 其特征在于， 该基因在提 高植物抗逆性中的应用。
PCT/CN2012/000306 2012-02-23 2012-03-12 枣树谷胱甘肽过氧化物酶基因 WO2013123625A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 201210042136 CN102533809B (zh) 2012-02-23 2012-02-23 枣树谷胱甘肽过氧化物酶基因
CN201210042136.X 2012-02-23

Publications (1)

Publication Number Publication Date
WO2013123625A1 true WO2013123625A1 (zh) 2013-08-29

Family

ID=46341840

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/000306 WO2013123625A1 (zh) 2012-02-23 2012-03-12 枣树谷胱甘肽过氧化物酶基因

Country Status (2)

Country Link
CN (1) CN102533809B (zh)
WO (1) WO2013123625A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103255153B (zh) * 2013-04-27 2014-09-24 山西省农业科学院果树研究所 枣树2-半胱氨酸氧化还原酶基因
CN108913670A (zh) * 2018-07-06 2018-11-30 浙江海洋大学 厚壳贻贝谷胱甘肽过氧化物酶--一种新型的海洋污染检测标记物
CN111826361B (zh) * 2020-07-30 2021-08-10 上海市农业科学院 香菇谷胱甘肽过氧化物酶LeGPX在提高微生物耐胁迫能力中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101381733A (zh) * 2007-09-03 2009-03-11 河南大学 拟南芥谷胱甘肽过氧化物酶基因had1在植物抗干旱方面应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101292998A (zh) * 2008-05-27 2008-10-29 沈荣法 一种蝇蛆粉的抗疲劳、抗衰老和抗肥胖保健功能方面的用途

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101381733A (zh) * 2007-09-03 2009-03-11 河南大学 拟南芥谷胱甘肽过氧化物酶基因had1在植物抗干旱方面应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 21 February 2007 (2007-02-21), KOH, C.S. ET AL.: "Populus trichocarpa x Populus deltoids glutathione peroxidase 5 (GPX5) Mrna, partial cds", accession no. F418010 *
DATABASE GENBANK 4 December 2009 (2009-12-04), TUSKAN, G.A. ET AL.: "Populus trichocarpa glutathione peroxidase (PtrcGpxS), mRNA", accession no. M_002299499 *
HU, MAOLONG ET AL.: "Glutathione peroxidise gene BnGPXl cloning from rapeseed and expression under abiotic stress", CHINESE JOURNALIST OF OIL CROP SCIENCES, vol. 33, no. 4, 31 August 2011 (2011-08-31), pages 331 - 337 *
ZHAO, ZHU: "Study on cloning and function of ThGPX2 and ThGPX6 genes of Thellungiella halophila", CHINA MASTER'S THESES FULL-TEXT DATABASE, vol. 1, 15 January 2009 (2009-01-15), pages 1 - 65 *
ZHOU, LIJING ET AL.: "Bioinformatics Analysis of Glutathione Peroxidase in Arabidopsis", JOURNAL OF MINZU UNIVERSITY OF CHINA (NATURAL SCIENCES EDITION), vol. 19, no. 2, 31 May 2010 (2010-05-31), pages 11 - 17 *

Also Published As

Publication number Publication date
CN102533809B (zh) 2013-03-20
CN102533809A (zh) 2012-07-04

Similar Documents

Publication Publication Date Title
CN107936104B (zh) 牡丹PsMYB12转录因子及其编码基因与应用
CN110791523B (zh) 一种棉花抗旱相关基因GhRCHY1及其应用
CN113604490B (zh) 猕猴桃溃疡病感病基因AcBXL1及其应用
RU2684719C2 (ru) Белок, ассоциированный с устойчивостью к болезни, и кодирующий его ген и их применение в регулировании устойчивости растений к болезни
CN110317250B (zh) Myb6基因及其编码蛋白在调控植物对黄萎病抗性中的应用
CN110904122A (zh) 一种苹果抗旱基因MdbHLH130及其应用
CN112898391B (zh) 枳抗寒基因PtrERF9在植物抗寒遗传改良中的应用
CN111454972B (zh) 枳抗寒基因PtrBADH及其在植物抗寒遗传改良中的应用
CN110452291A (zh) 铁皮石斛胚胎发育晚期丰富蛋白DoLEA43在促进植物愈伤组织形成中的应用
CN110229818B (zh) 蜡梅CpSNAC1基因启动子及其应用
CN113307853B (zh) 一种黑果枸杞花青素合成相关的MYB转录抑制因子LrMYB3及其应用
CN101880674B (zh) 枣树水通道蛋白基因及在提高植物耐旱性和耐盐性中的应用
WO2013123625A1 (zh) 枣树谷胱甘肽过氧化物酶基因
CN109837297B (zh) 与黄萎病抗性相关的GhAGD13基因及其应用
CN110862998A (zh) 紫苏fad8基因及其在提高植物不饱和脂肪酸含量和抗寒性能中的应用
CN108588088B (zh) 一种抗旱转录因子PbrERF109及其制备方法、应用和编码的蛋白质及应用
CN113929755B (zh) 一种葡萄霜霉菌分泌的植物免疫激活蛋白及引物和应用
CN113373161B (zh) GhERF017基因在调控植物耐盐性中的应用
CN108823220A (zh) 一种苹果中蜡质合成相关基因MdCER1的克隆及其应用
CN116606358A (zh) GmTLP8蛋白及其编码基因在调控植物耐逆性中的应用
CN114507674A (zh) 茶树昼夜节律基因lux在提高植物抗寒性上的应用
CN111171128A (zh) 番茄SlSWEET5b基因在防御南方根结线虫中的应用
CN114908068B (zh) 梨胼胝质合成酶PbrCalS5及其编码基因和应用
CN114685631B (zh) 樟疫霉效应子蛋白scr93258及其应用
NL2031934B1 (en) Proanthocyanidin transport-related gst protein gene in brown cotton fiber and use thereof

Legal Events

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

Ref document number: 12869444

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: 12869444

Country of ref document: EP

Kind code of ref document: A1