东北林蛙瘦素受体叠加转录蛋白基因的克隆及组织表达分析

Cloning and tissue expression analysis of the LepROT gene of Rana dybowskii

摘要:

瘦素受体叠加转录蛋白（leptin receptor overlapping transcript，LepROT）在免疫调节中发挥多种作用。为了获得两栖类LepROT在抗感染中的作用及其机制的信息，首先采用RT-PCR技术克隆东北林蛙的LepROT基因并进行生物信息学分析；再分别构建嗜水气单胞菌（Aeromonas hydrophila，Ah）与细菌脂多糖（lipopolysaccharides，LPS）东北林蛙的感染模型，获得组织病理解剖变化特征，并应用实时荧光定量逆转录聚合酶链反应（qRT-PCR）检测LepROT基因的变化规律及其对核转录因子NF-kB信号通路的影响。结果表明，LepROT基因长度为396 bp，编码131个氨基酸残基，与两栖类同源性在93.74%以上，与哺乳类同源性在86.39%以上，在各个物种中相对保守；感染Ah、LPS后，基因LepROT、NF-kB、IKKα和IKKβ的相对表达量均显著上调（P<0.01），并且不同组织的表达量上调的时间规律不同，推测东北林蛙可能通过LepROT启动NF-kB信号通路发挥抗感染作用，为进一步扩展两栖类LepROT的生物学功能的研究提供了证据。

关键词:LepROT基因;NF-kB信号通路;嗜水气单胞菌;细菌脂多糖;东北林蛙

Abstract:

Leptin receptor overlapping transcript (LepROT) plays multiple roles in the regulation of immune systems. However, very little information is available about the anti-infectious mechanisms of amphibians LepROT. In this study, the cDNA sequence of the Rana dybowskii LepROT gene was determined by using RT-PCR and bioinformatics analysis. Then, the Aeromonas hydrophila (Ah) and lipopolysaccharides (LPS) infected models of R. dybowskii was constructed to obtain histopathological characteristics. Constitutive expression of LepROT mRNA and NF-κB signaling pathway were detected by real-time quantitative PCR. The full-length cDNA of LepROT gene was 396 bp and encoded 131 amino acids. Amino acid sequence analysis revealed LepROT shares 93.74% and 86.39% identity with homologues from other amphibians and mammals respectively, and the LepROT gene was quite conserved among different species. After infection, the relative expression levels of LepROT, NF-kB, IKKα and IKKβ mRNA were all significantly upregulated (P<0.01), but showed a diverse temporal pattern of up-regulation in different tissues. Therefore, it was proposed that the LepROT gene of R. dybowskii might activate the NF-κB signaling pathway to exert anti-infectious effects, thus providing evidence for further extending the biological function of LepROT.

Key words:LepROT;NF-kB signaling pathway;Aeromonas hydrophila;lipopolysaccharides;Rana dybowskii