甜菜夜蛾几丁质脱乙酰酶SeCDA2a的外源表达及酶活力测定
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摘要
【目的】为了研究甜菜夜蛾几丁质脱乙酰酶SeCDA2a的酶学性质,探索其对甜菜夜蛾幼虫发育过程的影响,为甜菜夜蛾的生物防治提供新靶标。【方法】以pMD19-T-Secda2a重组质粒为模板,PCR扩增得到Secda2a基因。构建原核表达载体pET-28a-Secda2a,IPTG诱导蛋白表达。利用Bac-to-Bac杆状病毒表达系统,构建重组转座载体pFastBac HT A-Secda2a,脂质体转染昆虫细胞Sf9,Western blot对SeCDA2a重组蛋白进行分析,以对硝基苯胺为底物利用分光光度计测定其酶活力。【结果】Secda2a在大肠杆菌和Sf9中均成功表达61kDa的重组蛋白,与预测分子量大小相符。IPTG终浓度为0.50mmol/L、37℃诱导培养4h时蛋白表达量最高。昆虫细胞Sf9表达的重组蛋白SeCDA2a的酶活力是1.57U/mL。【结论】本研究实现甜菜夜蛾几丁质脱乙酰酶基因Secda2a的外源表达,测定重组蛋白SeCDA2a的酶活力。
【Objective】To study the enzymatic characterization of SeCDA2a fromSpodoptera exigua,and explore its effect on larval development,thus provide new targets for the biological control of S exigua.【Methods】The full-length cDNA of Secda2a was amplified with recombinant plasmid pMD19-T-Secda2a as PCR template.The prokaryotic expression vector pET-28 a-Secda2a was constructed and induced by IPTG in E.coli.The baculovirus expression vector pFastBac HT A-Secda2a was constructed using the Bac-to-Bac baculovirus expression system and transfected into insect cells Sf9 using liposome to obtain the recombinant protein SeCDA2a.Western blot was used to detect the recombinant protein.The enzyme activity was tested by spectrophotometer using p-nitroaniline as substrate.【Results】The Secda2a was successfully expressed as an expected molecular weight of 61 kDa in both E.coli and Sf9.The fusion protein was dominantly expressed when the hosts were cultured at inducing temperature of 37℃ for 4 hwith 0.50 mmol/L IPTG.The enzyme activity of recombinant protein SeCDA2a expressed by Sf9 was 1.57 U/mL.【Conclusion】The Secda2a gene was successfully expressed in vitro,and enzymatic characterization of recombinant protein acquired from Sf9 was analyzed in this study.
【Objective】To study the enzymatic characterization of SeCDA2a fromSpodoptera exigua,and explore its effect on larval development,thus provide new targets for the biological control of S exigua.【Methods】The full-length cDNA of Secda2a was amplified with recombinant plasmid pMD19-T-Secda2a as PCR template.The prokaryotic expression vector pET-28 a-Secda2a was constructed and induced by IPTG in E.coli.The baculovirus expression vector pFastBac HT A-Secda2a was constructed using the Bac-to-Bac baculovirus expression system and transfected into insect cells Sf9 using liposome to obtain the recombinant protein SeCDA2a.Western blot was used to detect the recombinant protein.The enzyme activity was tested by spectrophotometer using p-nitroaniline as substrate.【Results】The Secda2a was successfully expressed as an expected molecular weight of 61 kDa in both E.coli and Sf9.The fusion protein was dominantly expressed when the hosts were cultured at inducing temperature of 37℃ for 4 hwith 0.50 mmol/L IPTG.The enzyme activity of recombinant protein SeCDA2a expressed by Sf9 was 1.57 U/mL.【Conclusion】The Secda2a gene was successfully expressed in vitro,and enzymatic characterization of recombinant protein acquired from Sf9 was analyzed in this study.
引文
[1]司升云,周利琳,王少丽,江幸福,许再福,慕卫,王冬生,王小平,陈浩涛,杨亦桦,吉训聪.甜菜夜蛾防控技术研究与示范[J].应用昆虫学报,2012,49(6):1432-1438
[2]姜艳杰,谭伟明.甜菜夜蛾抗药性研究进展[J].安徽化工,2007,33(5):1-4
[3]Kramer K J&Koga D.Insect chitin:physical state,synthesis,degradation and metabolic regulation[J].Insect Biochemistry and Molecular Biology,1986,16(6):851-87
[4]Qu M&Yang Q.A novel alternative splicing site of class Achitin synthase from the insect Ostrinia furnacalis-Gene organization,expression pattern and physiological significance[J].Insect Biochemistry and Molecular Biology,2011,41(12):923-931
[5]Tsigos L,Martinou A,Kafetzopoulos D&Bouriotis V.Chitin deacetylases:new,versatile tools in biotechnology[J].Trends in Biotechnology,2000,18(7):305-312
[6]Guo W,Li G,Pang Y&Wang P.A novel chitin-binding protein identified from the peritrophic membrane of the cabbage looper,Trichoplusia ni[J].Insect Biochemistry and Molecular Biology,2005,35(11):1224-1234
[7]Toprak U,Baldwin D,Eelandson M,Gillott C,Hou X,Coutu C&Hegedus D D.A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata(Lepidoptera:Noctuidae)peritrophic matrix[J].Insect Molecular Biology,2008,17(5):573-585
[8]Zhong X W,Wang X H,Tan X,Xia Q Y,Xiang Z H&Zhao P.Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane[J].International Journal of Molecular Sciences,2014,15(2):1946-1961
[9]孙晓彤,赵丹,闫晓平,徐大庆,李少雅,李景.甜菜夜蛾几丁质脱乙酰酶1(SeCDA1)在毕赤酵母中的表达与活性测定[J].中国生物防治学报,2015,31(4):586-591
[10]闫晓平,赵丹,孙晓彤,郭巍,李少雅,李景.美国白蛾几丁质脱乙酰酶1(HcCDA1)的克隆表达与酶活测定[J].中国生物防治学报,2015,31(3):423-427
[11]赵盼,张学尧,刘晓健,赵小明,于荣荣,董玮,马恩波,张建珍,张敏.飞蝗几丁质脱乙酰基酶的真核表达、亲和纯化及酶活性[J].中国农业科学,2017,50(6):1057-1066
[12]刘丽萍,赵祥颖,刘建军,田延军,张家祥.一种简易、高效产几丁质脱乙酰酶菌种的筛选方法[J].食品与发酵工业,2008,34(1):65-68
[13]ZhaoY,Park R D&Muzzarelli R A A.Chitin Deacetylases:Properties and Applications[J].Marine Drugs,2010,8(1):24-46
[14]杨倩,刘建辉,蒋彤,董莉莉,段宏玉,孙纪录.几丁质脱乙酰酶的研究进展[J].食品研究与开发,2017,38(10):200-203
[15]Arakane Y,Dixit R,Begum K,Park Y,Specht C A,Merzendorfer H,Kramer K J,Muthukrishnan S&Beeman R W.Analysis of functions of the chitin deacetylase gene family in Tribolium castaneum[J].Insect Biochemistry and Molecular Biology,2009,39(5-6):355-365
[16]Xi Y,Pan P L,Ye Y X&Zhang C X.Chitin deacetylase family genes in the brown planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)[J].Insect Molecular Biology,2014,23(6):695-705
[17]于荣荣,丁国伟,郭亚平,马恩波,张建珍.中华稻蝗几丁质脱乙酰基酶基因的分子特性和生物学功能[J].中国农业科学,2014,47(7):1321-1329
[18]Yu R R,Liu W M,Li D Q,Zhao X M,Ding G W,Zhang M,Ma E B,Zhu K Y,Li S,Moussian B&Zhang J Z.Helicoidal organization of chitin in the cuticle of the migratory locust requires the function of the chitin deacetylase2enzyme(LmCDA2)[J].Journal of Biological Chemistry,2016,291(47):24352
[19]Quan G,Ladd T,Duan J,Wen F,Doucet D,Cusson M&Krell P J.Characterization of a spruce budworm chitin deacetylase gene:stage-and tissue-specific expression,and inhibition using RNA interference[J].Insect Biochemistry and Molecular Biology,2013,43(8):683-691
[20]Jakubowska A K,Caccia S,Gordon K H,Ferre J&Herrero S.Down-regulation of a chitin deacetylase-like protein in response to baculovirus infection and its application for improving baculovirus infectivity[J].Journal of Virology,2010,84(5):2547-2555
[21]孙晓彤.甜菜夜蛾几丁质脱乙酰酶1(CDA1)的鉴定与分析[D].保定:河北农业大学,2015:25-28
[22]闫晓平,赵丹,郭巍,王伟,张雅昆,郜玉杰,赵坤莉.美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质[J].中国农业科学,2017,50(5):849-858
[2]姜艳杰,谭伟明.甜菜夜蛾抗药性研究进展[J].安徽化工,2007,33(5):1-4
[3]Kramer K J&Koga D.Insect chitin:physical state,synthesis,degradation and metabolic regulation[J].Insect Biochemistry and Molecular Biology,1986,16(6):851-87
[4]Qu M&Yang Q.A novel alternative splicing site of class Achitin synthase from the insect Ostrinia furnacalis-Gene organization,expression pattern and physiological significance[J].Insect Biochemistry and Molecular Biology,2011,41(12):923-931
[5]Tsigos L,Martinou A,Kafetzopoulos D&Bouriotis V.Chitin deacetylases:new,versatile tools in biotechnology[J].Trends in Biotechnology,2000,18(7):305-312
[6]Guo W,Li G,Pang Y&Wang P.A novel chitin-binding protein identified from the peritrophic membrane of the cabbage looper,Trichoplusia ni[J].Insect Biochemistry and Molecular Biology,2005,35(11):1224-1234
[7]Toprak U,Baldwin D,Eelandson M,Gillott C,Hou X,Coutu C&Hegedus D D.A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata(Lepidoptera:Noctuidae)peritrophic matrix[J].Insect Molecular Biology,2008,17(5):573-585
[8]Zhong X W,Wang X H,Tan X,Xia Q Y,Xiang Z H&Zhao P.Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane[J].International Journal of Molecular Sciences,2014,15(2):1946-1961
[9]孙晓彤,赵丹,闫晓平,徐大庆,李少雅,李景.甜菜夜蛾几丁质脱乙酰酶1(SeCDA1)在毕赤酵母中的表达与活性测定[J].中国生物防治学报,2015,31(4):586-591
[10]闫晓平,赵丹,孙晓彤,郭巍,李少雅,李景.美国白蛾几丁质脱乙酰酶1(HcCDA1)的克隆表达与酶活测定[J].中国生物防治学报,2015,31(3):423-427
[11]赵盼,张学尧,刘晓健,赵小明,于荣荣,董玮,马恩波,张建珍,张敏.飞蝗几丁质脱乙酰基酶的真核表达、亲和纯化及酶活性[J].中国农业科学,2017,50(6):1057-1066
[12]刘丽萍,赵祥颖,刘建军,田延军,张家祥.一种简易、高效产几丁质脱乙酰酶菌种的筛选方法[J].食品与发酵工业,2008,34(1):65-68
[13]ZhaoY,Park R D&Muzzarelli R A A.Chitin Deacetylases:Properties and Applications[J].Marine Drugs,2010,8(1):24-46
[14]杨倩,刘建辉,蒋彤,董莉莉,段宏玉,孙纪录.几丁质脱乙酰酶的研究进展[J].食品研究与开发,2017,38(10):200-203
[15]Arakane Y,Dixit R,Begum K,Park Y,Specht C A,Merzendorfer H,Kramer K J,Muthukrishnan S&Beeman R W.Analysis of functions of the chitin deacetylase gene family in Tribolium castaneum[J].Insect Biochemistry and Molecular Biology,2009,39(5-6):355-365
[16]Xi Y,Pan P L,Ye Y X&Zhang C X.Chitin deacetylase family genes in the brown planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)[J].Insect Molecular Biology,2014,23(6):695-705
[17]于荣荣,丁国伟,郭亚平,马恩波,张建珍.中华稻蝗几丁质脱乙酰基酶基因的分子特性和生物学功能[J].中国农业科学,2014,47(7):1321-1329
[18]Yu R R,Liu W M,Li D Q,Zhao X M,Ding G W,Zhang M,Ma E B,Zhu K Y,Li S,Moussian B&Zhang J Z.Helicoidal organization of chitin in the cuticle of the migratory locust requires the function of the chitin deacetylase2enzyme(LmCDA2)[J].Journal of Biological Chemistry,2016,291(47):24352
[19]Quan G,Ladd T,Duan J,Wen F,Doucet D,Cusson M&Krell P J.Characterization of a spruce budworm chitin deacetylase gene:stage-and tissue-specific expression,and inhibition using RNA interference[J].Insect Biochemistry and Molecular Biology,2013,43(8):683-691
[20]Jakubowska A K,Caccia S,Gordon K H,Ferre J&Herrero S.Down-regulation of a chitin deacetylase-like protein in response to baculovirus infection and its application for improving baculovirus infectivity[J].Journal of Virology,2010,84(5):2547-2555
[21]孙晓彤.甜菜夜蛾几丁质脱乙酰酶1(CDA1)的鉴定与分析[D].保定:河北农业大学,2015:25-28
[22]闫晓平,赵丹,郭巍,王伟,张雅昆,郜玉杰,赵坤莉.美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质[J].中国农业科学,2017,50(5):849-858