飞蝗几丁质脱乙酰基酶的真核表达、亲和纯化及酶活性

doi:10.3864/j.issn.0578-1752.2017.06.007

摘要/Abstract

摘要： 【目的】体外真核表达飞蝗（Locusta migratoria）几丁质脱乙酰基酶1和2（chitin deacetylase 1 and 2，LmCDA1和LmCDA2）并测定其酶活性，为进一步明确飞蝗LmCDA1和LmCDA2在几丁质降解途径中的生理功能及研发新型绿色环保杀虫剂提供依据。【方法】使用BLASTP和SMART软件在线预测LmCDA1、LmCDA2a和LmCDA2b的结构域；PCR克隆获得目的基因LmCDA1、LmCDA2a和LmCDA2b的全长序列，并分别构建pFastBac-LmCDAs重组质粒，转化获得Bacmid重组质粒后，转染至昆虫Sf9细胞进行目的蛋白的体外表达。采用Western blot技术对目的蛋白表达情况进行检测，并通过Ni-NTA亲和层析柱和阴离子（Q-Sepharose）交换层析柱对蛋白产物进行纯化。12% SDS-PAGE检测蛋白纯度后，采用分光光度法以对硝基乙酰苯胺为底物检测目的蛋白的酶活性，T检验法对LmCDA2a和LmCDA2b酶活力进行差异显著性分析。【结果】BLASTP和SMART软件预测结果显示LmCDA1、LmCDA2a和LmCDA2b均含有4个结构域：N-端信号肽（signal peptide）、几丁质结合域（chitin binding peritrophin-A，ChBD）、A型低密度脂蛋白受体结构域（low-density lipoprotein receptor class A，LDLa）和脱乙酰基酶催化结构域（catalytic domain，CDA）。3个基因的几丁质结合域中均包含6个保守的半胱氨酸。LmCDA2a和LmCDA2b两个剪切子除在其第3个半胱氨酸和第4个半胱氨酸之间（67—84 aa）的氨基酸数目和组成及在第4和第6个半胱氨酸（84—106 aa）之间的序列存在差异外，其余部分完全一致。Western blot结果显示LmCDA1、LmCDA2a和LmCDA2b的蛋白分子量约为61 kD左右，与预测的蛋白分子量大小一致，表明Bacmid重组质粒在昆虫Sf9细胞中成功表达。采用12% SDS-PAGE胶电泳对各蛋白纯化组分进行检测，结果显示Ni-NTA亲和层析柱可将大部分杂蛋白洗脱，而Q-Sepharose交换层析柱可对蛋白进行更彻底地纯化。酶活检测结果显示LmCDA1、LmCDA2a和LmCDA2b的酶活力分别为0.268、0.354、0.228 U·μL^-1，并且LmCDA2a和LmCDA2b的酶活力存在显著差异。【结论】体外真核表达LmCDA1、LmCDA2a和LmCDA2b蛋白并进行酶活测定后发现三者均具有几丁质脱乙酰基酶活力，且LmCDA2a和LmCDA2b的酶活力具有显著性差异，推测前期研究中沉默LmCDA2a和LmCDA2b后分别出现不同飞蝗表型的原因可能是由于它们酶活力存在显著性差异。

关键词: 飞蝗, 几丁质脱乙酰基酶, 真核表达, 蛋白纯化, 酶活检测

Abstract: 【Objective】 The objective of this study is to investigate the eukaryotic expression and enzyme activity of chitin deacetylase 1 and 2 (LmCDA1 and LmCDA2) in Locusta migratoria. The results will provide an experimental basis to further clarify the physiological function of LmCDA1 and LmCDA2 in chitin degradation pathway and the development of new green pesticides.【Method】The domains of LmCDA1, LmCDA2a and LmCDA2b were predicted using BLASTP and SMART softwares. The full-length sequences of LmCDA1, LmCDA2a and LmCDA2b were obtained by PCR. Recombinant plasmids of pFastBac-LmCDAs were constructed, the recombinant Bacmid plasmids were obtained by transformation, and then transfected into Sf9 insect cells to express target proteins in vitro. Target proteins were detected by Western blot technology, and then purified by Ni-NTA affinity chromatography column and anion exchange chromatography column (Q-Sepharose). The purity of proteins was detected by 12% SDS-PAGE, and the enzymes activity was detected by spectrophotometric method with p-nitroacetylaniline as substrate. The method of T test was used to analyze the difference of enzyme activity of LmCDA2a and LmCDA2b. 【Result】 The results by using BLASTP and SMART softwares showed LmCDA1, LmCDA2a and LmCDA2b contained four structural domains, that are signal peptide, chitin binding peritrophin-A (ChBD), low-density lipoprotein receptor class A (LDLa) and catalytic domain (CDA). Three genes contained six conserved cysteines in ChBD. The spacing and amino acid composition between cysteines 3 and 4 (67-84 aa) and the sequence between cysteines 4 and 6 (84-106 aa) differed between LmCDA2a and LmCDA2b, the rest were identical. Western blot result showed that the protein molecular weight of LmCDA1, LmCDA2a and LmCDA2b was about 61 kD, consistent with the prediction, which suggested that the recombinant Bacmid plasmids were expressed successfully in Sf9 insect cells. The purity of purified proteins was detected by 12% SDS-PAGE electrophoresis. Most impurities could be removed by Ni-NTA affinity chromatography, and the proteins were further purified by Q-Sepharose exchange chromatography. Enzyme activity determination showed that LmCDA1, LmCDA2a and LmCDA2b had chitin deacetylase activity of 0.268, 0.354 and 0.228 U·μL^-1, respectively, and the activity of LmCDA2a and LmCDA2b showed significant differences. 【Conclusion】Eukaryotic expression in vitro and enzyme activity determination of LmCDA1, LmCDA2a and LmCDA2b revealed that these three enzymes have chitin deacetylase activity, and LmCDA2a and LmCDA2b enzyme activity has a significant difference. It was speculated that the reasons of different phenotypes of L. migratoria when LmCDA2a and LmCDA2b were silenced might be that LmCDA2a and LmCDA2b enzyme activity had a significant difference.

Key words: Locusta migratoria, chitin deacetylase, eukaryotic expression, protein purification, activity detection

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.

ZHAO Pan, ZHANG XueYao, LIU XiaoJian, ZHAO XiaoMing, YU RongRong, DONG Wei, MA EnBo, ZHANG JianZhen, ZHANG Min. Eukaryotic Expression, Affinity Purification and Enzyme Activity of Chitin Deacetylase in Locusta migratoria[J]. Scientia Agricultura Sinica, 2017, 50(6): 1057-1066.

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参考文献

[1] Zhang J Z, Liu X J, Zhang J Q, Li D Q, Sun Y, Guo Y P, Ma E B, Zhu K Y. Silencing of two alternative splicing-derived mRNA variants of chitin synthase 1 gene by RNAi is lethal to the oriental migratory locust, Locusta migratoria manilensis (Meyen). Insect Biochemistry and Molecular Biology,2010, 40(11): 824-833.

[2] 杨红军, 王东升, 张立顺, 谢建军. 东亚飞蝗对马拉硫磷抗性研究初报. 植保技术与推广, 2002, 22(8): 11-12.

Yang H J, Wang D S, Zhang L S, Xie J J. Preliminary study on the resistance of Locusta migratoria manilensis to malathion. Plant Protection Technology and Extension, 2002, 22(8): 11-12. (in Chinese)

[3] Yang M L, Zhang J Z, Zhu K Y, Xuan T, Liu X J, Guo Y P, Ma E B. Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen). Archives of Insect Biochemistry and Physiology, 2009, 71(1): 3-15.

[4] Peneff C, Mengin-Lecreulx D, Bourne Y. The crystal structures of Apo and complexed Saccharomyces cerevisiae GNA1 shed light on the catalytic mechanism of an amino-sugar N-acetyltransferase. The Journal of Biological Chemistry, 2001, 276(19): 16328-16334.

[5] 李大琪, 杜建中, 张建琴, 郝耀山, 刘晓健, 王亦学, 马恩波, 张建珍, 孙毅. 东亚飞蝗几丁质酶家族基因的表达特性与功能研究. 中国农业科学, 2011, 44(3): 485-492.

Li D Q, Du J Z, Zhang J Q, Hao Y S, Liu X J, Wang Y X, Ma E B, Zhang J Z, Sun Y. Study on expression characteristics and functions of chitinase family genes from Locusta migratoria manilensis (Meyen). Scientia Agricultura Sinica, 2011, 44(3): 485-492. (in Chinese)

[6] Araki Y, Ito E. A pathway of chitosan formation in Mucor rourii: enzymatic deacetylation of chitin. European journal of biochemistry, 1975, 55(1): 71-78.

[7] Guo W, Li G X, Pang Y, Wang P. A novel chitin-binding protein identified from the peritrophic membrane of the cabbage looper, Trichoplusia ni. Insect Biochemistry and Molecular Biology, 2005, 35(11): 1224-1234.

[8] Luschnig S, Batz T, Armbruster K, Krasnow M A. Serpentine and vermiform encode matrix proteins with chitin-binding and deacetylation domains that limit tracheal tube length in Drosophila. Current biology, 2006, 16(2): 186-194.

[9] Dixit R, Arakane Y, Specht C A, Richard C, Kramer K J, Beeman R W, Muthukrishnan S. Domain organization and phylogenetic analysis of proteins from the chitin deacetylase gene family of Tribolium castaneum andthree other species of insects. Insect Biochemistry and Molecular Biology, 2008, 38(4): 440-451.

[10] Toprak U, Baldwin D, Erlandson 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. Insect Molecular Biology, 2008, 17(5): 573-585.

[11] Campbell P M, Cao A T, Hines E R, East P D, Gordon K H. Proteomic analysis of the peritrophic matrix from the gut of the caterpillar, Helicoverpa armigera. Insect Biochemistry and Molecular Biology, 2008, 38(10): 950-958.

[12] 郝威, 何旭玲, 徐豫松. 家蚕几丁质脱乙酰基酶基因结构及mRNA选择性剪切与表达差异的研究. 蚕业科学, 2010, 36(6): 921-929.

Hao W, He X L, Xu Y S. Gene structure, mRNA alternative splicing and expression pattern of chitin deacetylases in silkworm, Bombyx mori. Science of sericulture, 2010, 36(6): 921-929. (in Chinese)

[13] Quan X G, Ladd T, Jun D, Wen F Y, Dourcet D, Cusson M, Krell P J. Characterization of a spruce budworm chitin deacetylase gene: stage-and tissue-specific expression, and inhibition using RNA interference. Insect Biochemistry and Molecular Biology, 2013, 43(8): 683-691.

[14] 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. Insect Biochemistry and Molecular Biology, 2009, 39(5/6): 355-365.

[15] 丁国伟, 于荣荣, 杨美玲, 马恩波, 杨静, 张建珍. 中华稻蝗几丁质脱乙酰基酶1基因的分子特性及功能. 昆虫学报, 2014, 57(11): 1265-1271.

Ding G W, Yu R R, Yang M L, Ma E B, YANG J, Zhang J Z. The molecular characterization and functional analysis of chitin deacetylase 1 gene in Oxya chinensis. Acta Entomologica Sinica, 2014, 57(11): 1265-1271. (in Chinese)

[16] 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2基因的分子特性和生物学功能. 中国农业科学, 2014, 47(7): 1321-1329.

Yu R R, Ding G W, Guo Y P, Ma E B, Zhang J Z. Molecular characterization and functional analysis of chitin deacetylase 2 gene in Oxya chinensis. Scientia Agricultura Sinica, 2014, 47(7): 1321-1329. (in Chinese)

[17] 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 deacetylase2 enzyme (LmCDA2). The Journal of Biological Chemistry, 2016, 291(47): 24352-24363.

[18] 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. International Journal of Molecular Sciences, 2014, 15(2): 1946-1961.

[19] 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. 食品与发酵工业, 2008, 34(1): 65-70.

Liu L P, Zhao X Y, Liu J J, Tian Y J, Zhang J X. A simple and efficient production of chitin deacetylase strains method. Food and Fermentation Industries, 2008, 34(1): 65-70. (in Chinese)

[20] Liu L, Song H F, Zhang L, Fan X T, Zhang Q, Chen K, Chen H Q, Zhou Y J. Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase. Archives of virology, 2013, 158(12): 2453-2463.

[21] Kauss H, Jeblick W, Young D H. Chitin deacetylase from the plant pathogen colletotrichum lindemuthianum. Plant Science Letters, 1983, 28(2): 231-236.

[22] Kafetzopoulos D, Thireos G, Voumakis J N, Bouriotis V. The primary structure of a fungal chitin deacetylase reveals the function for two bacterial gene products. Proceedings of the National Academy of Sciences of the United States of America, 1993, 90(17): 8005-8008.

[23] 田曙光, 王璇琳, 刘至玄, 章扬培, 宫锋. 四唑蓝比色法测定肝素酶活性. 军事医学, 2006, 30(1): 65-67.

Tian S G, Wang X L, Liu Z X, Zhang Y P, Gong F. Analysis of heparanase activity by colorimetry. Military Medical Sciences, 2006, 30(1): 65-67. (in Chinese)

[24] 肖丽霞, 师明磊, 王洋, 张彦, 沈文龙, 李德彬, 赵玉军, 赵志虎. NADH依赖型酶活性检测方法的建立. 中国生物工程杂志, 2012, 32(4): 72-75.

Xiao L X, Shi M L, Wang Y, Zhang Y, Shen W L, Li D B, Zhao Y J, Zhao Z H. An efficient and economic method for detecting the activity of NADH dependent enzyme. China Biotechnology, 2012, 32(4): 72-75. (in Chinese)

[25] Tokura S. Latest development on the application of chitin and chitosan. Kobunshi, 1995, 44(3): 112-115.

[26] 宋慧芳, 李应龙, 马恩波, 张建珍. 飞蝗β-N-乙酰氨基葡萄糖苷酶基因的表达及酶学特性分析. 中国农业科学, 2016, 49(21): 4140-4148.

SONG H F, LI Y L, MA E B, ZHANG J Z. The heterogenous expression and enzymatic characteristics of β-N-acetylglucosaminidase from Locusta migratoria. Scientia Agricultura Sinica, 2016, 49(21): 4140-4148. (in Chinese)