家蚕BmMet2基因的组织定位与激素诱导表达分析

The Tissue Localization and Hormone-induced Expression of BmMet2 in Silkworm,Bombyx mori

Met作为保幼激素(JH)的受体,能编码bHLH-PAS结构域结合DNA并调控下游基因的表达。为了探究BmMet2是否作为JH的受体参与调控家蚕的生长发育,以家蚕为材料,用生物信息学方法分析了BmMet2蛋白结构,利用实时定量PCR检测其在翅原基不同时期和激素诱导下的表达情况;原核表达了BmMet2中能与DNA结合的蛋白区域BmMet2DBD,并制备了抗体;利用蛋白质印迹法(Western blotting)和免疫组化检测了BmMet2在家蚕多个时期和各组织中的定位表达情况。结果显示:在翅原基中,BmMet2在5龄第3天和5龄第6天有较高水平的表达,蛹期的表达量较低,这与家蚕体内JH的滴度一致;BmMet2在5龄第6天、吐丝期和预蛹期的胸节表皮、脂肪体及翅原基等组织中都有较高的表达;JH和20E均对BmMet2有不同程度的诱导,说明其可能同时参与了JH和20E的调控,进而参与调控家蚕变态发育过程。

Met,as the receptor of Juvenile Hormone(JH),can encode the bHLH-PAS domain to bind to DNA and regulate the expression of downstream genes.In order to explore whether BmMet2 acts as a receptor of JH involved in regulating silkworm growth and development,Bombyx mori was used as the material,the bioinformatics methods were used to analyze the structure of BmMet2 protein,and real-time PCR was used to detect its expression in different periods of wing discs and hormonal induction.The protein region BmMet2DBD in BmMet2 that can bind to DNA was cloned and prokaryotically expressed and was used to prepare antibody.The Western blotting and immunohistochemistry were used to detect the localization and expression of BmMet2 in multiple periods and in various tissues.The results showed that BmMet2 had a higher expression level at 3 days and 6 days of the 5th instar in the wing discs and lower expression at the pupal stage,which is consistent with the JH titer in the silkworm.BmMet2 proteins were expressed in the thoracic epidermis,fat body and wing discs at day 6 of the 5th instar larvae,the wandering and prepupa stages,and the expression level was high.Both JH and 20E could induce the expression of BmMet2,indicating that it may be involved in the regulation of JH and 20E at the same time and participate in the regulation of the metamorphic development of the silkworm.