明小花蝽卵黄原蛋白的鉴定及表达分析

Identification and Expression of Vitellogenin in Oriusnagaii

本研究对明小花蝽Orius nagaii的卵黄原蛋白(vitellogenin,Vg)进行结构分析、原核表达、抗体制备以及时空动态表达分析,旨在为昆虫卵黄原蛋白的研究提供理论依据。用RACE方法克隆出OnVg基因全长,运用生物信息网站分析OnVg蛋白得出其具有3个保守结构域:脂蛋白N端结构域、未知功能结构域以及血管性血友病因子结构域。构建OnVg的原核表达载体pET-32a-c(+)-OnVg,转至大肠杆菌进行诱导表达,通过免疫昆明小鼠制备了anti-OnVg抗体。Western blot结果显示anti-OnVg抗体与OnVg蛋白能够特异性结合。qPCR检测Vg基因在明小花蝽若虫、成虫阶段和成虫不同组织中的时空动态表达,结果表明Vg基因在若虫阶段最低,成虫阶段表达量升高且3 d时表达水平达到最高;成虫腹部表达量最高,其次是肠道和卵巢。本研究结果可为探究明小花蝽的生殖调控机理提供理论支持。

In this study,we analyzed the structure,prokaryotic expression,antibody preparation and spatio-temporal dynamic expression of vitellogenin(Vg)from Orius nagaii,aiming to provide theoretical bases for study of Vg in insects.The full length of OnVg gene was cloned by RACE method,and the OnVg protein was analyzed by biological information website which had three conserved domains including lipoprotein N-terminal domain,unknown function domain and von Willebrand factor domain.The prokaryotic expression vector of OnVg,pET-32a-c(+)-OnVg,was constructed and transferred into Escherichia coli to induce expression.Antibody of anti-OnVg was prepared by immunizing Kunming mice.Western blot results showed that anti-OnVg antibody could bind specifically to OnVg protein.The spatio-temporal dynamic expression of Vg gene in stages of nymphs,adults,and different adult tissues were detected by qPCR,and the results showed that the expression level of Vg gene was the lowest in nymphs,but increased in adults,and reached the highest level on the 3rd day of adult.In adults,it expressed the highest in abdomen,followed by intestine and ovary.The results of this study could provide theoretical supports for exploring the mechanism of reproductive regulation of O.nagaii.