东北林蛙adipor1和adipor2基因克隆及其在感染中的表达分析

Cloning of adipor1 and adipor2 genes in Rana dybowskii and its expression pattern upon infection

摘要:

脂联素受体1 (adiponectin receptor 1, AdipoR1)和脂联素受体2 (adiponectin receptor 2, AdipoR2)能够与脂肪组织分泌的脂联素(adiponectin, AdipoQ)相结合，参与机体的多种生理功能。为探究AdipoR1和AdipoR2在嗜水气单胞菌(Aeromonas hydrophila, Ah)感染下两栖类炎症反应中的作用，通过逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)技术克隆东北林蛙(Rana dybowskii)的adipor1和adipor2基因，对adipor1和adipor2进行生物信息学分析；利用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, qRT-PCR)技术分析adipor1和adipor2的组织表达差异，构建Ah感染的东北林蛙炎症模型，通过苏木精-伊红染色法(hematoxylin-eosin staining, HE)观察组织病理变化；再利用qRT-PCR和Western blotting对感染后不同时间点的adipor1和adipor2的表达进行动态检测。结果表明，AdipoR1和AdipoR2均为具有7次跨膜结构域的膜蛋白；系统进化树也显示与两栖类聚为同一分支；基于qRT-PCR和Western blotting结果显示，Ah感染下adipor1和adipor2在转录和翻译水平发生不同程度的上调表达，但应答时间和水平有所差异。综上所述，推测AdipoR1和AdipoR2参与了机体的细菌免疫应答过程，为进一步探究脂联素受体在两栖类的生物学功能提供参考。

Abstract:

Adiponectin receptor 1 (AdipoR1) and Adiponectin receptor 2 (AdipoR2) can bind to adiponectin (AdipoQ) secreted by adipose tissue to participate in various physiological functions of the body. In order to explore the role of AdipoR1 and AdipoR2 in amphibians infected by Aeromonas hydrophila (Ah), the genes adipor1 and adipor2 of Rana dybowskii were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by bioinformatics. The tissue expression difference of adipor1 and adipor2 was analyzed by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and an inflammatory model of R. dybowskii infected by Ah was constructed. The histopathological changes were observed by hematoxylin-eosin staining (HE staining); the expression profiles of adipor1 and adipor2 after infection were dynamically detected by qRT-PCR and Western blotting. The results show that AdipoR1 and AdipoR2 are cell membrane proteins with seven transmembrane domains. Phylogenetic tree also shows that AdipoR1 and AdipoR2 cluster with the amphibians in the same branch. qRT-PCR and Western blotting results show that adipor1 and adipor2 were up-regulated at different levels of transcription and translation upon Ah infection, but the response time and level were different. It is speculated that AdipoR1 and AdipoR2 participate in the process of bacterial immune response, providing a basis for further exploring the biological functions of AdipoR1 and AdipoR2 in amphibians.