中国农业科学 ›› 2017, Vol. 50 ›› Issue (5): 849-858.doi: 10.3864/j.issn.0578-1752.2017.05.008

• 植物保护 • 上一篇    下一篇

美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质

闫晓平1,赵丹1,郭巍1,2,王伟1,张雅昆1,郜玉杰1,赵坤莉1

 
  

  1. 1河北农业大学植物保护学院,河北保定071000;2北京农学院植物科学技术学院,北京102206
  • 收稿日期:2016-10-12 出版日期:2017-03-01 发布日期:2017-03-01
  • 通讯作者: 郭巍,E-mail:1787421502@qq.com
  • 作者简介:闫晓平,E-mail:1013479394@qq.com。
  • 基金资助:
    国家自然科学基金(31471775)、国家现代农业(花生)产业技术体系(30971910)、北京市百千万人才工程项目

Cloning, Expression and Enzymatic Characterization of Chitin Deacetylases from Hyphantria cunea

YAN Xiaoping1, ZHAO Dan1, GUO Wei1,2, WANG Wei1, ZHANG YaKun1, GAO YuJie1, ZHAO KunLi1   

  1. 1College of Plant Protection, Agricultural University of Hebei, Baoding 071000, Hebei; 2Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206
  • Received:2016-10-12 Online:2017-03-01 Published:2017-03-01

摘要: 【目的】研究美国白蛾(Hyphantria cunea)几丁质脱乙酰酶(chitin deacetylase,CDA)基因的酶学性质,了解其在昆虫生命过程中如何发挥功能,为美国白蛾的生物防治提供新靶标。【方法】对家蚕(Bombyx mori)、棉铃虫(Helicoverpa armigera)和云杉卷夜蛾(Choristoneura fumiferana)3种鳞翅目昆虫的几丁质脱乙酰酶2序列保守域进行分析,采用同源比对方法,设计几丁质脱乙酰酶2基因特异引物,利用PCR扩增该基因全长cDNA序列;构建原核重组表达载体pET30a-HcCDA2,克隆获得编码美国白蛾HcCDA2的基因,IPTG诱导蛋白表达,SDS-PAGE电泳检测;构建重组杆状病毒表达载体pFastBac-HcCDA1 和pFastBac-HcCDA2,脂质体转染法转染昆虫细胞Hi5,分别获得P1、P2、P3病毒,收集P3病毒上清,得到美国白蛾HcCDA1(前期研究所得)和HcCDA2重组蛋白,Western blot分析;重组蛋白HcCDA1和HcCDA2经硫酸铵粗纯化后,以对硝基乙酰苯胺为底物,测定重组几丁质脱乙酰酶HcCDA1&2的酶活力,对其最适反应温度、最适pH以及金属离子的影响等酶学性质进行研究。【结果】 获得了编码美国白蛾几丁质脱乙酰酶2基因全长cDNA序列,命名为HcCDA2 (GenBank登录号:KT781841),基因全长1.6 kb,该基因在大肠杆菌中成功表达61 kD目的蛋白,免疫家兔获得HcCDA2蛋白的特异性多克隆抗体。Western blot分析结果表明,HcCDA1和HcCDA2在昆虫细胞(Hi5)中均成功表达约80 kD蛋白。酶学性质研究表明,昆虫细胞中分泌表达的HcCDA1和HcCDA2均具有催化活性,两种酶的最适反应温度均为50℃,当温度达到80℃时,HcCDA2几乎失去酶活力;HcCDA1酶促反应适宜的pH范围为7.0—9.0,并且HcCDA1和HcCDA2酶促反应的最适pH均为8.0;在最适反应条件下,HcCDA2蛋白酶活力均高于HcCDA1蛋白酶活力;Mg2+、Zn2+、Mn2+和Ca2+对HcCDA1和HcCDA2酶促反应整体呈抑制趋势,随浓度增大,Zn2+对HcCDA1抑制作用逐渐增强,而Mg2+对HcCDA1的抑制作用则呈现先升高后降低的趋势,而且Mg2+和Ca2+对HcCDA2的抑制作用也是呈现先升高后降低的趋势。Co2+和Fe2+对HcCDA1和HcCDA2酶促反应有激活作用,随浓度增大,Fe2+激活作用越强,而Co2+激活作用则呈现先升高后降低的趋势。【结论】克隆了编码美国白蛾几丁质脱乙酰酶2基因(HcCDA2),在原核细胞中表达61 kD目的蛋白,免疫家兔获得HcCDA2蛋白的特异性抗体。得到具有活性的美国白蛾几丁质脱乙酰酶HcCDA1和HcCDA2,两者在体外均检测到催化活性,两种酶的最适反应温度均为50℃,最适pH均为8.0。

关键词: 美国白蛾, 几丁质脱乙酰酶, 克隆表达, 酶学性质

Abstract: 【Objective】 The objective of this paper is to study the function of the enzyme in the process of insect life, enzymatic characterization of chitin deacetylases from Hyphantria cunea and to provide a basis for understanding of its function in the insect life, and to provide new targets for the biological control strategy of H. cunea. 【Method】 The conserved domain of chitin deacetylase 2 sequences from three kinds of lepidoptera insects Bombyx mori, Helicoverpa armigera and Choristoneura fumiferana were analyzed and chitin deacetylase gene-specific primers were designed by homologous alignment. A cDNA clone was identified to contain cDNA sequence coding for chitin deacetylase by PCR amplification. The prokaryotic recombinant expression vector pET30a-HcCDA2 was constructed and cloned in E. coli, the protein expression was induced by IPTG and detected by SDS-PAGE electorphoresis. The recombinant baculovirus expression vector pFastBac-HcCDA1 and pFastBac-HcCDA2 were constructed, and liposome transfection method was used to transfect insect cells Hi5, then the P1, P2 and P3 virus were obtained, respectively. The P3 cells supernatant were collected and western blot analysis was applied to detect the chitin deacetylase 1 (from the previous study) and 2 protein expression. The recombinant proteins were crude purified with ammonium sulfate and then the enzymatic characterizations were studied with the 4’-nitroacetanilide (including the optimum temperature, the optimum pH and the effects of different metal ions on enzyme activity).【Result】 The full-length cDNA clone encoded chitin deacetylase 2 in H. cunea was identified (GenBank accession number: KT781841). The cDNA is 1.6 kb in length. The protein encoded by this cDNA is named HcCDA2 and the protein was shown a band on the SDS-PAGE gel with an apparent molecular weight of 61 kD. The specific antibodies reacting to HcCDA2 were obtained by immunizing rabbits. TheHcCDA1 and HcCDA2 were shown as 80 kD proteins in Hi5 cell line detected by Western blot analysis. The study of enzymatic properties showed that the two enzymes which were secreting expression in insect cells possessed catalytic activity, and the optimum temperature were both 50℃, when the temperature reached 80℃, the HcCDA2 almost lost its activity; HcCDA1 enzymatic reaction appropriate pH ranged from 7.0-9.0, and the optimum pH of HcCDA1 and HcCDA2 were both 8.0. Under the optimum reaction conditions, the enzyme activities of HcCDA2 protein were higher than HcCDA1 protein. Mg2+, Zn2+, Mn2+ and Ca2+ showed an inhibition on HcCDA1 and HcCDA2, an increasing Zn2+ concentration would lead to the enhancement of inhibitory effects on HcCDA1, but Mg2+ showed a trend of increasing first and then decreasing on HcCDA1, as well as an increasing Mg2+ or Ca2+ concentration showed an inhibition on HcCDA2. Co2+ and Fe2+ showed activation on HcCDA1 and HcCDA2, Fe2+ showed activation stably with increasing concentration, while Co2+ showed a trend of increasing first and then decreasing. 【Conclusion】 The gene HcCDA2 was cloned from H. cunea, expressed in E. coli shown as a 61 kD protein, and the HcCDA2 protein-specific polyclonal antibodies were obtained by immunizing rabbits. The active HcCDA1 and HcCDA2 proteins were obtained, and both of them showed a catalytic activity in vitro, the optimum temperature and pH were both 50℃ and 8.0, respectively.

Key words: Hyphantria cunea, chitin deacetylase, cloning and expression, enzymatic characterization