东亚飞蝗Locusta migratoria manilensis(Meyen)是我国主要的农业害虫之一,已发现东亚飞蝗对某些农药产生了抗性,其抗性机制可能与谷胱甘肽硫转移酶(GST)代谢解毒相关。本研究利用特异性引物合成东亚飞蝗GST4个不同家族基因的双链RNA(dsRNA),将dsRNA注射到东亚飞蝗幼虫体内,采用Real time RT-PCR技术测定了干扰不同时间后目的基因mRNA的表达水平。结果表明,4个不同家族GST的沉默效应具有时间差异。来自delta家族的LmGSTd1和sigma家族的LmGSTs5基因在注射dsRNA后12h时mRNA量就已显著下降;而来自theta家族的LmGSTt1和unknown家族的LmGSTu1基因在注射24h后mRNA水平才呈现显著下降。本研究对后续东亚飞蝗GST功能及抗性机制研究提供了基础资料和依据,同时对其它昆虫RNA干扰研究具有一定的借鉴作用。
A cDNA encoding a sigma-class glutathione S-transferase of the locust, Locusta migratoria manilensis (LmGSTs1), was cloned by reverse transcriptase-polymerase chain reaction. The 830 bp-long cDNA encoded a 615 bp open reading frame (204 amino acid polypeptide), which exhibited the structural motif and domain organization characteristic of GST sigma-class. It revealed 59, 57, 57, and 56% identities to sigma-class GSTs from Blattella germanica, Gryllotalpa orientalis, Nasonia vitripennis, and Pediculus humanus corporis, respectively. A recombinant protein (LmGSTs1) was functionally expressed in Escherichia coli cells in a soluble form and purified to homogeneity. LmGSTs1 was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GSTs, as well as with p-nitro-benzyl chloride. Its optimal activity was observed at pH 8.0 and at 30℃. Incubation for 30 min at temperatures below 50℃ scarcely affected the activity. The I50 of reactive blue (RB) was 18.5 μmol L-1. In the presence of 0.05 mmol L-1 ethacrynic acid (ECA), LmGSTs1 showed (81±3)% of the original activities.
