关键词: 茶尺蠖, 无机焦磷酸酶, 原核表达, 功能验证, qRT-PCR

Abstract: 【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 (LC₁₀=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 1 279.6 U/mg, and the optimum temperature, pH and metal ion of enzymatic reaction were 65℃, pH 7.5, and Mg²⁺, 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.

Key words: Ectropis obliqua, inorganic pyrophosphatase, prokaryotic expression, functional verification, qRT-PCR