水芹HSP90基因的原核表达及条件优化
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摘要
植物热激蛋白90 (HSP90)在响应高温胁迫中起到重要作用。本研究从水芹中克隆到HSP90基因(基因登录号:MF961800),将其连接到原核表达载体pET30a上,构建重组质粒pET30a-OjHSP90。对大肠杆菌菌株BL21表达重组蛋白的起始菌液浓度、IPTG浓度、诱导温度和诱导时间进行了优化。结果显示,OjHSP90基因在大肠杆菌中可实现诱导表达,目的蛋白分子质量约为80 kD,与预期结果相符。当重组菌的起始OD600值达到0.8时表达量最高,pET30a-OjHSP90的最佳IPTG诱导浓度为0.2 mmol/L,最佳诱导温度为37℃,最适诱导时间为4 h。本研究通过诱导条件的优化可以使目的蛋白的表达量显著增加,以期为研究OjHSP90的耐热功能提供理论依据。
The plant heat shock protein 90(HSP90) plays important roles in response to high temperature stress.The HSP90 gene(Gene Bank No.MF961800) was cloned from Oenanthe javanica in this study, and then inserted into the prokaryotic expressing vector pET30 a to construct the recombinant plasmid pET30 a-OjHSP90. The growth conditions of E. coli strain BL21 expressing OjHSP90 including the induced start concentration, IPTG concentration, induction temperature and time were optimized. The results showed that the molecular weight of target protein was about 80 kD, which was consistent with the expected results. The optimal bacterium and ITPG concentration, induced temperature and time were OD600=0.8, 0.2 mmol/L, 37℃ and 4 h, respectively. The expression of target protein was significantly increased by conditions optimization. This study will provide theoretical foundation for further research of OjHSP90 thermotolerance.
The plant heat shock protein 90(HSP90) plays important roles in response to high temperature stress.The HSP90 gene(Gene Bank No.MF961800) was cloned from Oenanthe javanica in this study, and then inserted into the prokaryotic expressing vector pET30 a to construct the recombinant plasmid pET30 a-OjHSP90. The growth conditions of E. coli strain BL21 expressing OjHSP90 including the induced start concentration, IPTG concentration, induction temperature and time were optimized. The results showed that the molecular weight of target protein was about 80 kD, which was consistent with the expected results. The optimal bacterium and ITPG concentration, induced temperature and time were OD600=0.8, 0.2 mmol/L, 37℃ and 4 h, respectively. The expression of target protein was significantly increased by conditions optimization. This study will provide theoretical foundation for further research of OjHSP90 thermotolerance.
引文
Csermely P.,Schnaider T.,Soti C.,Prohaszka Z.,and Nardai G.,1998,The 90-kDa molecular chaperone family:structure,function,and clinical applications,a comprehensive review,Pharmacol.Ther.,79(2):129-168
Dai Z.Z.,Li B.,Xu Y.,Gi D.H.,Chen C.C.,and Xie C.T.,2014,Cloning and expression pattern analysis of two heat shock protein(Hsp90)genes from Pyropia haitanensis,Shuichan Xuebao(Journal of Fisheries of China),38(3):340-349(代真真,李兵,徐燕,纪德华,陈昌生,谢潮添,2014,坛紫菜两种Hsp90基因的克隆及表达特征分析,水产学报,38(3):340-349)
Hu Y.P.,Li D.M.,Zhu B.B.,Huang W.F.,Zhou M.,Cai X.L.,and Wang M.,2016,Cloning and bioinformation analysis of OjHSP90 gene from cress(Oenanthe javanica),Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(11):2959-2963(胡艳平,李德明,朱白婢,黄文枫,周曼,蔡兴来,王敏,2016,水芹OjHSP90基因克隆及生物信息学分析,分子植物育种,14(11):2959-2963)
Hubert D.A.,Tornero P.,Belkhadir Y.,Krishna P.,Takahashi A.,Shirasu K.,and Dang J.L.,2003,Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein,EMBO J.,22(21):5679-5689
Kregel K.C.,2002,Heat shock proteins:modifying factors in physiological stress responses and acquired thermotolerance,J.Appl.Physiol.,92(5):2177-2186
Li L.,Xu Z.S.,Liu L.,Yu Z.,Li L.C.,Chen M.,and Ma Y.Z.,2009,Expression of wheat heat shock protein WHSP90 gene and preparation of its polyclonal antibodies,Mailei Zuowu Xuebao(Journal of Triticeae Crops),29(3):380-384(李琳,徐兆师,刘丽,于卓,李连城,陈明,马有志,2009,小麦热激蛋白WHSP90基因的原核表达及多克隆抗体制备,麦类作物学报,29(3):380-384)
Liao Y.C.,Xu Y.H.,Zhang Z.,and Wei J.H.,2016,Prokaryotic expression and purification of Aquilaria sinensis(Lour.)Gilg As MYC2 protein,Yaoxue Xuebao(Acta Pharmaceutica Sinica),51(4):662-667(廖永翠,徐艳红,张争,魏建和,2016,白木香As MYC2蛋白的原核表达与纯化,药学学报,51(4):662-667)
Lindquist S.,and Craig E.A.,1988,The heat-shock proteins,Annu.Rev.Genet.,22(3):631-677
Liu Z.,Zhang L.,Pu Y.,Liu Z.,Li Z.,Zhao Y.,and Qin S.,2014,Cloning and expression of a cytosolic HSP90 gene in Chlorella vulgaris,Biomed Res.Int.,2014:487050
Mclellan C.A.,Turbyville T.J.,Wijeratne E.M.K.,Kerschen A.,Vierling E.,Queitsch C.,Whitesell L.,and Gunatilaka A.A.L.,2007,A rhizosphere fungus enhances Arabidopsis thermotolerance through production of an HSP90 inhibitor,Plant Physiol.,145(1):174-182
Picard D.,2002,Heat-shock protein 90,a chaperone for folding and regulation,Cell Mol.Life Sci.,59(10):1640-1648
Timperio A.M.,Egidi M.G.,and Zolla L.,2008,Proteomics applied on plant abiotic stresses:role of heat shock proteins(HSP),J.Proteomics,71(4):391-411
Wang L.H.,Jiang J.Z.,Deng J.J.,Jiang W.,and Chen T.,2013,Effects of density on yield and quality of heat-resistant water dropwort,Yangzhou Daxue Xuebao(Journal of Yangzhou University(Agricultural and Life Science Edition)),34(4):108-110(王丽慧,江解增,邓静娟,姜雯,陈亭,2013,不同移栽密度对耐热水芹产量和品质的影响,扬州大学学报(农业与生命科学版),34(4):108-110)
Wang N.,Gu S.Q.,Fan Y.S.,Li P.,Wang W.X.,and Dong J.G.,2010,Construction and expression of prokaryotic expression vector of STK1 from Setosphaeria turcica,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),43(18):3876-3881(王宁,谷守芹,范永山,李坡,王文秀,董金皋,2010,玉米大斑病菌STK1原核表达载体的构建及其表达,中国农业科学,43(18):3876-3881)
Wang W.,Vinocur B.,and Altman A.,2003,Plant responses to drought,salinity and extreme temperatures:towards genetic engineering for stress tolerance,Planta,218:1-14
Wang W.,Vinocur B.,Shoseyov O.,and Altman A.,2004,Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response,Trends Plant Sci.,9(5):244-252
Wang Y.,Jiang J.Z.,and Liu H.,2010,Preliminary studies on effects of solar-shading screen cover on quality of wettish-cultivated water dropwort(Oenanthe stolonifera(Roxb)Wall.),Zhongguo Shucai(China Vegetables),(6):70-73(王雁,江解增,刘浩,2010,遮阳网覆盖对湿栽水芹品质的影响初探,中国蔬菜,(6):70-73)
Yamada K.,Fukao Y.M.,Fukazawa M.,Suzuki I.,and Nishimura M.,2007,Cytosolic HSP90 regulates the heat shock response that is responsible for heat acclimation in Arabidopsis thaliana,J.Biol.Chem.,282(52):37794-37804
Dai Z.Z.,Li B.,Xu Y.,Gi D.H.,Chen C.C.,and Xie C.T.,2014,Cloning and expression pattern analysis of two heat shock protein(Hsp90)genes from Pyropia haitanensis,Shuichan Xuebao(Journal of Fisheries of China),38(3):340-349(代真真,李兵,徐燕,纪德华,陈昌生,谢潮添,2014,坛紫菜两种Hsp90基因的克隆及表达特征分析,水产学报,38(3):340-349)
Hu Y.P.,Li D.M.,Zhu B.B.,Huang W.F.,Zhou M.,Cai X.L.,and Wang M.,2016,Cloning and bioinformation analysis of OjHSP90 gene from cress(Oenanthe javanica),Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(11):2959-2963(胡艳平,李德明,朱白婢,黄文枫,周曼,蔡兴来,王敏,2016,水芹OjHSP90基因克隆及生物信息学分析,分子植物育种,14(11):2959-2963)
Hubert D.A.,Tornero P.,Belkhadir Y.,Krishna P.,Takahashi A.,Shirasu K.,and Dang J.L.,2003,Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein,EMBO J.,22(21):5679-5689
Kregel K.C.,2002,Heat shock proteins:modifying factors in physiological stress responses and acquired thermotolerance,J.Appl.Physiol.,92(5):2177-2186
Li L.,Xu Z.S.,Liu L.,Yu Z.,Li L.C.,Chen M.,and Ma Y.Z.,2009,Expression of wheat heat shock protein WHSP90 gene and preparation of its polyclonal antibodies,Mailei Zuowu Xuebao(Journal of Triticeae Crops),29(3):380-384(李琳,徐兆师,刘丽,于卓,李连城,陈明,马有志,2009,小麦热激蛋白WHSP90基因的原核表达及多克隆抗体制备,麦类作物学报,29(3):380-384)
Liao Y.C.,Xu Y.H.,Zhang Z.,and Wei J.H.,2016,Prokaryotic expression and purification of Aquilaria sinensis(Lour.)Gilg As MYC2 protein,Yaoxue Xuebao(Acta Pharmaceutica Sinica),51(4):662-667(廖永翠,徐艳红,张争,魏建和,2016,白木香As MYC2蛋白的原核表达与纯化,药学学报,51(4):662-667)
Lindquist S.,and Craig E.A.,1988,The heat-shock proteins,Annu.Rev.Genet.,22(3):631-677
Liu Z.,Zhang L.,Pu Y.,Liu Z.,Li Z.,Zhao Y.,and Qin S.,2014,Cloning and expression of a cytosolic HSP90 gene in Chlorella vulgaris,Biomed Res.Int.,2014:487050
Mclellan C.A.,Turbyville T.J.,Wijeratne E.M.K.,Kerschen A.,Vierling E.,Queitsch C.,Whitesell L.,and Gunatilaka A.A.L.,2007,A rhizosphere fungus enhances Arabidopsis thermotolerance through production of an HSP90 inhibitor,Plant Physiol.,145(1):174-182
Picard D.,2002,Heat-shock protein 90,a chaperone for folding and regulation,Cell Mol.Life Sci.,59(10):1640-1648
Timperio A.M.,Egidi M.G.,and Zolla L.,2008,Proteomics applied on plant abiotic stresses:role of heat shock proteins(HSP),J.Proteomics,71(4):391-411
Wang L.H.,Jiang J.Z.,Deng J.J.,Jiang W.,and Chen T.,2013,Effects of density on yield and quality of heat-resistant water dropwort,Yangzhou Daxue Xuebao(Journal of Yangzhou University(Agricultural and Life Science Edition)),34(4):108-110(王丽慧,江解增,邓静娟,姜雯,陈亭,2013,不同移栽密度对耐热水芹产量和品质的影响,扬州大学学报(农业与生命科学版),34(4):108-110)
Wang N.,Gu S.Q.,Fan Y.S.,Li P.,Wang W.X.,and Dong J.G.,2010,Construction and expression of prokaryotic expression vector of STK1 from Setosphaeria turcica,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),43(18):3876-3881(王宁,谷守芹,范永山,李坡,王文秀,董金皋,2010,玉米大斑病菌STK1原核表达载体的构建及其表达,中国农业科学,43(18):3876-3881)
Wang W.,Vinocur B.,and Altman A.,2003,Plant responses to drought,salinity and extreme temperatures:towards genetic engineering for stress tolerance,Planta,218:1-14
Wang W.,Vinocur B.,Shoseyov O.,and Altman A.,2004,Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response,Trends Plant Sci.,9(5):244-252
Wang Y.,Jiang J.Z.,and Liu H.,2010,Preliminary studies on effects of solar-shading screen cover on quality of wettish-cultivated water dropwort(Oenanthe stolonifera(Roxb)Wall.),Zhongguo Shucai(China Vegetables),(6):70-73(王雁,江解增,刘浩,2010,遮阳网覆盖对湿栽水芹品质的影响初探,中国蔬菜,(6):70-73)
Yamada K.,Fukao Y.M.,Fukazawa M.,Suzuki I.,and Nishimura M.,2007,Cytosolic HSP90 regulates the heat shock response that is responsible for heat acclimation in Arabidopsis thaliana,J.Biol.Chem.,282(52):37794-37804
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