苹果树腐烂病菌木聚糖酶VmXyl1基因的原核表达及多克隆抗体制备
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摘要
木聚糖酶(xylanase)基因VmXyl1在苹果树腐烂病菌—黑腐皮壳菌(Valsa mali)致病过程中发挥重要作用。为了揭示VmXyl1在腐烂病菌与寄主互作过程中的作用机制,本研究构建了原核表达载体pET-32a-VmXyl1,利用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside, IPTG)进行诱导,在大肠杆菌(Escherichia coli)表达菌株Rosetta (DE3)中成功表达了VmXyl1融合蛋白;利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)检测融合蛋白的可溶性和纯化效果,免疫新西兰白兔(Oryctolagus cuniculus)制备多克隆抗体,并对其效价和特异性进行分析。结果显示,该蛋白主要以包涵体形式存在,且0.5 mmol/L IPTG、30℃诱导8 h融合蛋白高效表达;经NiNTA柱亲和层析纯化后,获得了预期大小的融合蛋白。酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)和Western blot分析表明,所制备抗体效价达1∶102 400,且抗体能特异性识别VmXyl1融合蛋白及发病苹果(Malus pumila)树皮组织中的木聚糖酶蛋白,说明所制备的抗体具有高效性和特异性。研究结果为深入探究腐烂病菌木聚糖酶的致病机理及其与寄主的互作机制提供了基础资料。
Xylanase VmXyl1 plays an important role in the pathogenesis of Valsa mali. In order to reveal the action mechanism of VmXyl1 in the interaction of V. mali and host, a recombinant plasmid pET-32a-VmXyl1 was constructed in this study. The fusion protein VmXyl1 was expressed successfully in Escherichia coli Rosetta(DE3) strain when induced by isopropyl β-D-1-thiogalactopyranoside(IPTG). The solubility and purification effects of the fusion protein were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Then, New Zealand white rabbits(Oryctolagus cuniculus) were immunized with purified VmXyl1 protein and polyclonal antibody was prepared. The titer and specificity of the antibody wereanalyzed by enzyme-linked immunosorbent assay(ELISA) and Western blot. The sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) results showed that the fusion protein VmXyl1 mainly existed in the form of inclusion body, and the protein was expressed abundantly under induction by 0.5 mmol/L IPTG for 8 h at 30 ℃. The target protein with high purity was obtained by the Ni-NTA affinity chromatography. ELISA showed that the titer of prepared antibody was up to 1∶102 400. Western blot analysis demonstrated the polyclonal antibody could specifically recognize both the fusion protein VmXyl1 and xylanase protein in the apple(Malus pumila) bark tissue infected by V. mali, which indicated that the prepared antibody had high efficiency and specificity. The results in this study provide basic data for further exploring the pathogenesis of V. mali xylanase and its interaction mechanism with the host.
Xylanase VmXyl1 plays an important role in the pathogenesis of Valsa mali. In order to reveal the action mechanism of VmXyl1 in the interaction of V. mali and host, a recombinant plasmid pET-32a-VmXyl1 was constructed in this study. The fusion protein VmXyl1 was expressed successfully in Escherichia coli Rosetta(DE3) strain when induced by isopropyl β-D-1-thiogalactopyranoside(IPTG). The solubility and purification effects of the fusion protein were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Then, New Zealand white rabbits(Oryctolagus cuniculus) were immunized with purified VmXyl1 protein and polyclonal antibody was prepared. The titer and specificity of the antibody wereanalyzed by enzyme-linked immunosorbent assay(ELISA) and Western blot. The sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) results showed that the fusion protein VmXyl1 mainly existed in the form of inclusion body, and the protein was expressed abundantly under induction by 0.5 mmol/L IPTG for 8 h at 30 ℃. The target protein with high purity was obtained by the Ni-NTA affinity chromatography. ELISA showed that the titer of prepared antibody was up to 1∶102 400. Western blot analysis demonstrated the polyclonal antibody could specifically recognize both the fusion protein VmXyl1 and xylanase protein in the apple(Malus pumila) bark tissue infected by V. mali, which indicated that the prepared antibody had high efficiency and specificity. The results in this study provide basic data for further exploring the pathogenesis of V. mali xylanase and its interaction mechanism with the host.
引文
曹克强,国立耘,李保华,等.2009.中国苹果树腐烂病发生和防治情况调查[J].植物保护,35(2):114-116.(Cao KQ,Guo L Y,Li B H,et al.2009.Investigations on the occurrence and control of apple canker in China[J]Plant Protection,35(2):114-116.)
陈德鑫,李雯雯,李思斌,等.2017.烟草NteIF2α的原核表达、纯化及多克隆抗体制备和应用[J].农业生物技术学报,25(1):50-57.(Chen D X,Li W W,Li S B,et al2017.Prokaryotic expression,purification of NteIF2αand preparation and application of polyclonal antibody in Nicotiana tabacum[J].Journal of Agricultural Biotechnology,25(1):50-57.)
陈红歌,刘艳芳,刘亮伟,等.2009.木聚糖酶和木聚糖酶抑制蛋白在植物-病原菌互作中的作用[J].植物生理学通讯,45(2):176-182.(Chen H G,Liu Y F,Liu L L,e al.2009.Roles of endoxylanases and their inhibitors in plant-phytopathogen interaction[J].Plant Physiology Communications,45(2):176-182.)
陈晓林,牛程旺,李保华,等.2012.苹果树腐烂病菌产生细胞壁降解酶的种类及其活性分析[J].华北农学报,27(2):207-212.(Chen X L,Niu C W,Li B H,et al.2012The kinds and activities of cell wall-degrading enzymes produced by Valsa ceratosperma[J].Acta Agriculturae Boreali-Sinica,27(2):207-212.)
陈浙,宋革,周雪萍,等.2016.西瓜花叶病毒(WMV)单克隆抗体的制备及其应用[J].中国农业科学,49(14):2711-2724.(Chen Z,Song G,Zhou X P,et al.2016.Preparation and application of monoclonal antibodies against Watermelon mosaic virus(WMV)[J].Scientia Agricultura Sinica,49(14):2711-2724.)
董飞,祭芳,吴季荣,等.2013.单端孢酶烯3-O-乙酰转移酶编码基因Tri101的原核表达及多克隆抗体制备[J].中国农业科学,46(16):3369-3376.(Dong F,Ji F,Wu J R,et al.2013.Prokaryotic expression and polyclonal antibody preparation of trichothecene 3-O-acetyltransferase gene Tri101[J].Scientia Agricultura Sinica,46(16):3369-3376.)
黄传臻,刘香利,曹汝菲,等.2017.小麦CWI-B1的原核表达、纯化与多克隆抗体制备[J].农业生物技术学报,25(7):1102-1110.(Huang C Z,Liu X L,Cao R F,et al.2017.Prokaryotic expression,purification and preparation of polyclonal antibody for wheat(Triticum aestivum)CWI-B1[J].Journal of Agricultural Biotechnology,25(7):1102-1110.)
李保华,王彩霞,董向丽.2013.我国苹果主要病害研究进展与病害防治中的问题[J].植物保护,39(5):46-54.(Li B H,Wang C X,Dong X L.2013.Research progress in apple diseases and problems in the disease management in China[J].Plant Protection,39(5):46-54.)
李超,李保华,李桂舫,等.2014.苹果树腐烂病菌致病因子及其与菌株致病性的关系[J].北方园艺,2014(13):118-122.(Li C,Li B H,Li G F,et al.2014.Pathogenic factors produced by Valsa mali var.mali and their relationship with pathogenicity of different strains[J].Northern Horticulture,2014(13):118-122.)
史祥鹏.2016.苹果树腐烂病菌木聚糖酶基因的克隆及功能分析[D].硕士学位论文,青岛农业大学,导师:李保华,pp.7-16.(Shi X P.2016.Cloning and function analysis of xylanase genes of Valsa mali var.mali[D].Thesis for M.S.,Qingdao Agriculture University,Supervisor:Li BH,pp.7-16.)
王彩霞,王国平,洪霓,等.2006.柑橘衰退病毒多克隆和单克隆抗体的制备及检测效果分析[J].生物工程学报,22(4):629-634.(Wang C X,Wang G P,Hong N,et al.2006.Production of polyclonal and monoclonal antibodies against Citrus tristeza virus and their efficiency for the detection of the virus[J].Chinese Journal of Biotechnology,22(4):629-634.)
王彩霞,董向丽,张振芳,等.2012a.2011年烟台苹果产区腐烂病发病情况调查与原因分析[J].植物保护,38(3):136-138.(Wang C X,Dong X L,Zhang Z F,et al.2012Outbreak and the reasons of apple valsa canker in Yantai apple production area in 2011[J].Plant Protection,38(3):136-138.)
王彩霞,张清明,李桂舫,等.2012b.苹果树腐烂病拮抗细菌菌株BJ1的鉴定及其抑菌作用[J].植物保护学报,39(5):431-437.(Wang C X,Zhang Q M,Li G F,et al2012.Identification of the antagonistic bacteria BJ1 and its antifungal activity against Valsa ceratosperma[J].Acta Phytophylacica Sinica,39(5):431-437.)
王传振,刘晓鹏,崔怡,等.2016.猪转录因子Nanog高效表达、多克隆抗体制备及其应用[J].农业生物技术学报24(1):35-43.(Wang C Z,Liu X P,Cui Y,et al.2016High level expression,preparation and application of anti-nanog polyclonal antibody of porcine(Sus scrofa)Nanog protein[J].Journal of Agricultural Biotechnology,24(1):35-43.)
许春景,吴玉星,戴青青,等.2016.苹果树腐烂病菌多聚半乳糖醛酸酶基因Vmpg7和Vmpg8的功能[J].中国农业科学,49(8):1489-1498.(Xu C J,Wu Y X,Dai Q Q,e al.2016.Function of polygalacturonase genes Vmpg7and Vmpg8 of Valsa mali[J].Scientia Agricultura Sinica49(8):1489-1498.)
徐秋芳,陈晴晴,倪海平,等.2014.灰飞虱原肌球蛋白的基因克隆、原核表达及多克隆抗体制备[J].中国农业科学,47(19):3791-3798.(Xu Q F,Chen Q Q,Ni H P,e al.2014.Cloning,prokaryotic expression and polyclonal antibody preparation of tropomyosin gene in Laodelphax striatellus Fallén[J].Scientia Agricultura Sinica47(19):3791-3798.)
Annis S L,Goodwin P H.1997.Recent advances in the molecular genetics of plant cell wall-degrading enzymes produced by plant pathogenic fungi[J].European Journal of Plant Pathology,103(1):1-14.
Brito N,Espino J J,González C.2006.The endo-β-1,4-xylanase Xyn11A is required for virulence in Botrytis cinerea[J].Molecular Plant-Microbe Interactions,19(1):25-32.
Collins T,Gerday C,Feller G.2005.Xylanases,xylanase families and extremophilic xylanases[J].FEMS Microbiology Reviews,29(1):3-23.
Kubicek C P,Starr T L,Glass N L.2014.Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi[J].Annual Review of Phytopathology,52(1):427.
Lu J,Wei D,Wang Y,et al.2011.High-level expression and single-step purification of recombinant Bacillus anthracis protective antigen from Escherichia coli[J].Biotechnology and Applied Biochemistry,52(2):107-112.
Ma Z,Song T,Zhu L,et al.2015.A Phytophthora sojae glycoside hydrolase 12 protein is a major virulence factor during soybean infection and is recognized as a PAMP[J].Plant Cell,27(7):2057-2072.
Nguyen Q B,Itoh K,Van V B,et al.2011.Simultaneous silencing of endo-β-1,4 xylanase genes reveals their roles in the virulence of Magnaporthe oryzae[J].Molecular Microbiology,81(4):1008-1019.
Noda J,Brito N,González C.2010.The Botrytis cinerea xylanase Xyn11A contributes to virulence with its necrotizing activity,not with its catalytic activity[J].BMC Plant Biology,10(1):38.
Siah A,Deweera C,Duymec F,et al.2009.In planta xylanase activity and pathogenicity on wheat-mycosphaerella graminicola pathosystem[J].Communications in Agricultural and Applied Biological Sciences,74(3):693-700.
Xu M,Gao X,Chen J,et al.2017.The feruloyl esterase genes are required for full pathogenicity of the apple tree canker pathogen Valsa mali[J].Molecular Plant Pathology,19(6):1353-1363.
Yang Y,Yang X,Dong Y,et al.2018.The Botrytis cinerea Xylanase BcXyl1 modulates plant immunity[J].Frontiers in Microbiology,9:2535.
Yu C L,Li T,Shi X P,et al.2018.Deletion of endo-β-1,4-xylanase VmXyl1 impacts the virulence of Valsa mali in apple tree[J].Frontiers in Plant Science,9:663.
陈德鑫,李雯雯,李思斌,等.2017.烟草NteIF2α的原核表达、纯化及多克隆抗体制备和应用[J].农业生物技术学报,25(1):50-57.(Chen D X,Li W W,Li S B,et al2017.Prokaryotic expression,purification of NteIF2αand preparation and application of polyclonal antibody in Nicotiana tabacum[J].Journal of Agricultural Biotechnology,25(1):50-57.)
陈红歌,刘艳芳,刘亮伟,等.2009.木聚糖酶和木聚糖酶抑制蛋白在植物-病原菌互作中的作用[J].植物生理学通讯,45(2):176-182.(Chen H G,Liu Y F,Liu L L,e al.2009.Roles of endoxylanases and their inhibitors in plant-phytopathogen interaction[J].Plant Physiology Communications,45(2):176-182.)
陈晓林,牛程旺,李保华,等.2012.苹果树腐烂病菌产生细胞壁降解酶的种类及其活性分析[J].华北农学报,27(2):207-212.(Chen X L,Niu C W,Li B H,et al.2012The kinds and activities of cell wall-degrading enzymes produced by Valsa ceratosperma[J].Acta Agriculturae Boreali-Sinica,27(2):207-212.)
陈浙,宋革,周雪萍,等.2016.西瓜花叶病毒(WMV)单克隆抗体的制备及其应用[J].中国农业科学,49(14):2711-2724.(Chen Z,Song G,Zhou X P,et al.2016.Preparation and application of monoclonal antibodies against Watermelon mosaic virus(WMV)[J].Scientia Agricultura Sinica,49(14):2711-2724.)
董飞,祭芳,吴季荣,等.2013.单端孢酶烯3-O-乙酰转移酶编码基因Tri101的原核表达及多克隆抗体制备[J].中国农业科学,46(16):3369-3376.(Dong F,Ji F,Wu J R,et al.2013.Prokaryotic expression and polyclonal antibody preparation of trichothecene 3-O-acetyltransferase gene Tri101[J].Scientia Agricultura Sinica,46(16):3369-3376.)
黄传臻,刘香利,曹汝菲,等.2017.小麦CWI-B1的原核表达、纯化与多克隆抗体制备[J].农业生物技术学报,25(7):1102-1110.(Huang C Z,Liu X L,Cao R F,et al.2017.Prokaryotic expression,purification and preparation of polyclonal antibody for wheat(Triticum aestivum)CWI-B1[J].Journal of Agricultural Biotechnology,25(7):1102-1110.)
李保华,王彩霞,董向丽.2013.我国苹果主要病害研究进展与病害防治中的问题[J].植物保护,39(5):46-54.(Li B H,Wang C X,Dong X L.2013.Research progress in apple diseases and problems in the disease management in China[J].Plant Protection,39(5):46-54.)
李超,李保华,李桂舫,等.2014.苹果树腐烂病菌致病因子及其与菌株致病性的关系[J].北方园艺,2014(13):118-122.(Li C,Li B H,Li G F,et al.2014.Pathogenic factors produced by Valsa mali var.mali and their relationship with pathogenicity of different strains[J].Northern Horticulture,2014(13):118-122.)
史祥鹏.2016.苹果树腐烂病菌木聚糖酶基因的克隆及功能分析[D].硕士学位论文,青岛农业大学,导师:李保华,pp.7-16.(Shi X P.2016.Cloning and function analysis of xylanase genes of Valsa mali var.mali[D].Thesis for M.S.,Qingdao Agriculture University,Supervisor:Li BH,pp.7-16.)
王彩霞,王国平,洪霓,等.2006.柑橘衰退病毒多克隆和单克隆抗体的制备及检测效果分析[J].生物工程学报,22(4):629-634.(Wang C X,Wang G P,Hong N,et al.2006.Production of polyclonal and monoclonal antibodies against Citrus tristeza virus and their efficiency for the detection of the virus[J].Chinese Journal of Biotechnology,22(4):629-634.)
王彩霞,董向丽,张振芳,等.2012a.2011年烟台苹果产区腐烂病发病情况调查与原因分析[J].植物保护,38(3):136-138.(Wang C X,Dong X L,Zhang Z F,et al.2012Outbreak and the reasons of apple valsa canker in Yantai apple production area in 2011[J].Plant Protection,38(3):136-138.)
王彩霞,张清明,李桂舫,等.2012b.苹果树腐烂病拮抗细菌菌株BJ1的鉴定及其抑菌作用[J].植物保护学报,39(5):431-437.(Wang C X,Zhang Q M,Li G F,et al2012.Identification of the antagonistic bacteria BJ1 and its antifungal activity against Valsa ceratosperma[J].Acta Phytophylacica Sinica,39(5):431-437.)
王传振,刘晓鹏,崔怡,等.2016.猪转录因子Nanog高效表达、多克隆抗体制备及其应用[J].农业生物技术学报24(1):35-43.(Wang C Z,Liu X P,Cui Y,et al.2016High level expression,preparation and application of anti-nanog polyclonal antibody of porcine(Sus scrofa)Nanog protein[J].Journal of Agricultural Biotechnology,24(1):35-43.)
许春景,吴玉星,戴青青,等.2016.苹果树腐烂病菌多聚半乳糖醛酸酶基因Vmpg7和Vmpg8的功能[J].中国农业科学,49(8):1489-1498.(Xu C J,Wu Y X,Dai Q Q,e al.2016.Function of polygalacturonase genes Vmpg7and Vmpg8 of Valsa mali[J].Scientia Agricultura Sinica49(8):1489-1498.)
徐秋芳,陈晴晴,倪海平,等.2014.灰飞虱原肌球蛋白的基因克隆、原核表达及多克隆抗体制备[J].中国农业科学,47(19):3791-3798.(Xu Q F,Chen Q Q,Ni H P,e al.2014.Cloning,prokaryotic expression and polyclonal antibody preparation of tropomyosin gene in Laodelphax striatellus Fallén[J].Scientia Agricultura Sinica47(19):3791-3798.)
Annis S L,Goodwin P H.1997.Recent advances in the molecular genetics of plant cell wall-degrading enzymes produced by plant pathogenic fungi[J].European Journal of Plant Pathology,103(1):1-14.
Brito N,Espino J J,González C.2006.The endo-β-1,4-xylanase Xyn11A is required for virulence in Botrytis cinerea[J].Molecular Plant-Microbe Interactions,19(1):25-32.
Collins T,Gerday C,Feller G.2005.Xylanases,xylanase families and extremophilic xylanases[J].FEMS Microbiology Reviews,29(1):3-23.
Kubicek C P,Starr T L,Glass N L.2014.Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi[J].Annual Review of Phytopathology,52(1):427.
Lu J,Wei D,Wang Y,et al.2011.High-level expression and single-step purification of recombinant Bacillus anthracis protective antigen from Escherichia coli[J].Biotechnology and Applied Biochemistry,52(2):107-112.
Ma Z,Song T,Zhu L,et al.2015.A Phytophthora sojae glycoside hydrolase 12 protein is a major virulence factor during soybean infection and is recognized as a PAMP[J].Plant Cell,27(7):2057-2072.
Nguyen Q B,Itoh K,Van V B,et al.2011.Simultaneous silencing of endo-β-1,4 xylanase genes reveals their roles in the virulence of Magnaporthe oryzae[J].Molecular Microbiology,81(4):1008-1019.
Noda J,Brito N,González C.2010.The Botrytis cinerea xylanase Xyn11A contributes to virulence with its necrotizing activity,not with its catalytic activity[J].BMC Plant Biology,10(1):38.
Siah A,Deweera C,Duymec F,et al.2009.In planta xylanase activity and pathogenicity on wheat-mycosphaerella graminicola pathosystem[J].Communications in Agricultural and Applied Biological Sciences,74(3):693-700.
Xu M,Gao X,Chen J,et al.2017.The feruloyl esterase genes are required for full pathogenicity of the apple tree canker pathogen Valsa mali[J].Molecular Plant Pathology,19(6):1353-1363.
Yang Y,Yang X,Dong Y,et al.2018.The Botrytis cinerea Xylanase BcXyl1 modulates plant immunity[J].Frontiers in Microbiology,9:2535.
Yu C L,Li T,Shi X P,et al.2018.Deletion of endo-β-1,4-xylanase VmXyl1 impacts the virulence of Valsa mali in apple tree[J].Frontiers in Plant Science,9:663.