脊椎动物雌激素对家蚕卵黄原蛋白基因表达的调控及其与蜕皮激素受体相互作用的MST分析

Regulation of the expression of vitellogenin gene by vertebrate estrogen and a microscale thermophoresis assay of its interaction with ecdysone receptors in the silkworm,Bombyx mori ( Lepidoptera: Bombycidae)

【目的】雌激素雌二醇(E_2)在脊椎动物中通过雌激素受体(ER)调节脊椎动物的生长发育和繁殖。昆虫中并没有ER,但有研究显示某些昆虫能对E_2产生一定的应答。本研究在明确E_2能影响家蚕Bombyx mori卵黄原蛋白基因(BmVg)表达的前提下,分析其对这一基因表达的调控机制。【方法】取BmVg高量表达时的家蚕雌性幼虫(上蔟后60 h)脂肪体,用不同浓度(0.001,0.05,0.5,5和50 nmol/L)E_2处理后,通过qRT-PCR检测BmVg表达的变化,分析家蚕对E_2的应答;克隆家蚕蜕皮激素受体基因(BmEcRB1),构建原核表达载体,表达并纯化BmEcRB1蛋白后与E_2体外孵育,通过微量热泳动仪(MST)分析E_2与BmEcRB1的结合情况。【结果】不同浓度E_2均显著抑制离体培养的雌蚕脂肪体中BmVg基因的表达。MST实验证实,E_2与BmEcRB1具有一定的亲和力,解离常数(Kd)为77.8±22μmol/L。【结论】结果说明,E_2对BmVg表达的影响是通过与蜕皮激素(20E)竞争性结合BmEcRB1实现的。

[ Aim ] Estradiol （ E2 ）, an estrogen hormone, regulates the estrogen receptors （ERs） in vertebrates. There is no ER in insects, but can respond to E2. In this study, the possible regulatory mechanisms gene by E2 was elucidated in the silkworm, Bombyx mori, after co growth and reproduction through studies showed that some insects of the expression of vitellogenin nfirming that E2 influenced the expression of BmVg in female larvae of the silkworm. [ Methods ] The expression level of BmVg was detected in the fat body of female larvae （60 h after mounting） of B. mori after in vitro treatment with different concentrations of E2（0. 001, 0.05, 0. 5, 5 and 50 nmol/L） by qRT-PCR, and the response of the silkworm to E2 was analyzed. The ecdysone receptor gene of B. mori （BmEcRB1） was cloned and its prokaryotic expression vector was constructed. The BmEcRB1 protein was purified and incubated with E2in vitro, and the binding characteristics of E2 to BmEcRB1 were assayed by microscale thermophoresis （MST）. [ Results] Different concentrations of E2 significantly inhibited the expression of BmVg of female larvae of B. mori after in vitro culture, and the inhibitory effect was more obvious with the increase of E2 concentration. MST results showed that E2 had a certain affinity with BmEcRB1, with the dissociation constant （Kd） value of 77. 8 ± 22 μmol/L. [ Conclusion ] The results suggest that E2 inhibits the expression of BmVg in the silkworm through competition with ecdysone to bind BmEcRBI.