Expression Profiles Of Glutathione S-transferases (GSTs) Genes Of Bombyx Mori Induced By Insect Hormones

Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes in almost all living organisms, especially the role in the detoxification of xenobiotics, resistance to insect viruses and pesticides and protection against oxidative stress. Ecdysone (20-hydroxyecdysone) and juvenile hormone (JH) are main hormones in insects and play key role in the process of ecdysis, metamorphosis and production of yolk proteins. In order to understand the effect of these two insect hormones on GSTs (BmGSTD2 and BmGSTS2) expression, we used quantitative real time RT-PCR to examine expression profiles of Bombyx mori GST-Sigma (BmGSTS2) and GST-Delta (BmGSTD2) genes in the larval midgut of the silkworm after exposure to 2-hydroxyecdysone (20E) and juvenile hormone analogue (JHA) in this study. And we further analyzed the GSTs expression at the protein using column purification and LTQ mass spectrometry. To elucidate the ecdysone receptor (EcR) information in the promoter sequence of BmGSTD2 and BmGSTS2, we aligned the B. mori genomic sequence using the Gene Quest Module of DNASTAR, and demonstrated that there are two EcRs in the BmGSTD2 promoter and three in the BmGSTS2 promoter. The results suggest that insect hormone maybe play roles in regulating the expression of those genes.The results of real-time PCR indicated that expression of these two GSTs gene had dose-dependentent of insect hormones. In concentration-course study, 20E at higher concentrations (1.0μg/μL and 2.0μg/μL) caused significant upregulation of BmGSTD2, and all concentrations (0.5μg/μL to 2.0μg/μL) of 20E caused significant upregulation of BmGSTS2. However, JHA in all concentrations downregulated the expression of BmGSTD2 and BmGSTS2. When exposed to either 20E (2.0μg/μL) or JHA (2.0μg/μL) on the 3rd day of the fifth instar, the silkworm had higher BmGSTD2 at later time points: 15 h, 18 h and 24 h for 20E and 24 h for JHA. BmGSTS2 expression was downregulated within 24 h after exposure to JHA and showed a time-dependent response after exposure to 20E. We also did a stage-dependent study, in which JHA downregulated BmGSTD2 expression and upregulated BmGSTS2 expression significantly at both day 1 and day 3 of the fifth instar. 20E upregulated the expression of BmGSTD2 and BmGSTS2 at the two stages. These findings imply that hormones have an important role in the regulation of basal GST expression. These two GSTs gene expression are different after induced by insect hormone. This may be related to their different gene structure, regulatory elements and other factors. However, further validation and field trials should be carried out on the regulatory elements relevant to BmGSTD2 and BmGSTS2 gene expression.In order to know the effect of insect hormone on the expression of BmGSTD2 and BmGSTS2 at the level of post-transcription, we purified the GSTs protein through the reduced glutathione (GSH) pillar; and analyzed by SDS-PAGE. The result show that there are two bands in the graph with molecular weight range of 23-24kD. LTQ results indicated that the two bands are sigma-class GST (23339.74Da) and delta-class GST (24226.82Da), respectively. Protein Quantitation result showed that ether JHA or 20E (2.0μg/μL) downregulated BmGSTs protein expression.