茶尺蠖无机焦磷酸酶EoPPase672的表达与功能研究

Expression and functional analysis of inorganic pyrophosphatase EoPPase672 from Ectropis obliqua(Lepidoptera:Geometridae)

【目的】本研究旨在通过克隆茶尺蠖Ectropis obliqua解毒相关无机焦磷酸酶(inorganic pyrophosphatase,PPase)基因EoPPase672,并对其进行原核表达和功能验证,为更好地了解EoPPase672在茶尺蠖解毒过程中的作用及相关功能的应用提供理论依据。【方法】从茶尺蠖转录组数据库中筛选到EoPPase672 cDNA,构建原核表达载体pET-32a-EoPPase672,转化到大肠杆菌Escherichia coli BL21(DE3),SDS-PAGE及Western blot鉴定重组表达蛋白;测定EoPPase672的浓度以及酶促反应最适温度、最适pH和最适金属离子。利用qRT-PCR检测亚致死浓度(LC_(10)=2.08 mg/L)溴氰菊酯刺激后不同时间不同龄期茶尺蠖幼虫中EoPPase672的表达水平。基于PPase的特性,分析重组蛋白EoPPase672在PCR反应中对焦磷酸盐(PPi)的去除效果和对PCR扩增效率的影响。【结果】通过原核表达和纯化获得重组蛋白EoPPase672。通过无机磷酸盐(Pi)含量标准曲线测得该酶的比活力为1279.6 U/mg,酶促反应的最适温度为65℃、最适pH为7.5、最适金属离子为Mg^(2+)。茶尺蠖幼虫被溴氰菊酯(2.08 mg/L)刺激12 h后,2和3龄幼虫EoPPase672的表达量与对照组的相比显著上调,4龄幼虫EoPPase672的表达量小幅上调;被溴氰菊酯刺激24 h后,2和3龄幼虫EoPPase672的表达量与对照组的相比仍然显著上调,但与刺激12 h的2和3龄幼虫中的相比下调。在PCR反应中添加重组蛋白EoPPase672后,部分PPi被水解,解除了PPi对PCR扩增的抑制作用,对PCR扩增效率起到了增强效果。【结论】这些结果表明,重组蛋白EoPPase672可提高PCR扩增效率,EoPPase672基因可能参与了茶尺蠖的解毒过程。研究结果为进一步研究茶尺蠖EoPPase672的功能提供了依据。

【Aim】The objective of this study is to clone the coding sequence of the inorganic pyrophosphatase(PPase)gene EoPPase672 related to detoxification in the tea geometrid,Ectropis obliqua and to conduct prokaryotic expression and functional verification,so as to provide a theoretical basis for further understanding the role of EoPPase672 in the detoxification process of this insect and the application of related functions.【Methods】The EoPPase672 cDNA was screened from the transcriptome database of E.obliqua.The prokaryotic expression vector pET-32a-EoPPase672 was constructed and transformed into Escherichia coli BL21(DE3).The recombinant protein was identified by SDS-PAGE and Western blot,and its concentration and the optimum temperature,pH and metal ion in the enzymatic reaction were detected.The expression levels of EoPPase672 in E.obliqua larvae at different instars after exposed to the sublethal concentration(LC_(10)=2.08 mg/L)of deltamethrin for different time were determined by qRT-PCR.Based on the characteristics of PPase,the effects of the recombinant EoPPase672 in PCR reaction on the removal of pyrophosphate(PPi)and the PCR amplification efficiency were analyzed.【Results】The recombinant EoPPase672 was obtained by prokaryotic expression and purification.According to the standard curve of inorganic phosphate(Pi)content,the specific activity of the enzyme was 1279.6 U/mg,and the optimum temperature,pH and metal ion of enzymatic reaction were 65℃,pH 7.5,and Mg^(2+),respectively.After exposure to deltamethrin(2.08 mg/L)for 12 h,the expression levels of EoPPase672 in the 2nd and 3rd instar larvae were significantly up-regulated and that in the 4th instar larvae was slightly up-regulated as compared to that of the control group.After exposure to deltamethrin for 24 h,the expression levels of EoPPase672 in the 2nd and 3rd instar larvae were still significantly up-regulated as compared with that of the control group,but down-regulated as compared with those in the 2nd and 3rd instar larvae exposed to deltamethrin for 12 h.After adding the recombinant EoPPase672 in the PCR reaction,partial PPi was hydrolyzed,resulting in relieving the inhibition of PPi on PCR amplification and enhancing the PCR amplification efficiency.【Conclusion】These results show that the recombinant EoPPase672 can enhance the PCR amplification efficiency,and EoPPase672 may be involved in the detoxification process of E.obliqua,providing a basis for further studying the function of EoPPase672 in E.obliqua.