嗜水气单胞菌胁迫下东北林蛙MHCⅠ基因在不同组织的表达

Expression of MHCⅠ genes in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila

摘要:

本研究旨在探讨嗜水气单胞菌 (Aeromonas hydrophila，Ah) 胁迫下MHCⅠ基因在东北林蛙不同组织的表达特征，为揭示两栖类抗感染免疫应答机制提供依据。文中首先构建嗜水气单胞菌感染下的试验动物模型，通过苏木精-伊红染色法 (Hematoxylin-eosin staining，HE染色) 观察病理学变化；利用RT-PCR克隆东北林蛙MHCⅠ基因α1+α2肽结合区并进行生物信息学分析，再运用荧光定量PCR技术检测Ah胁迫下MHCⅠ在不同组织中的转录水平。结果表明：在Ah感染后，皮肤、肝脏和肌肉等组织均出现细胞结构消失和纹理紊乱等现象；获得MHCⅠ基因α1+α2肽结合区片段494 bp，可编码164个氨基酸，与两栖类同源性在77%以上，与哺乳类同源性低至14.96%，表明MHCⅠ基因α1+α2区在不同物种间保守性较低；荧光定量PCR结果显示，在Ah胁迫下，实验组肝脏、脾脏、肾脏、皮肤和肌肉组织MHCⅠ基因的转录水平在72 h前均高于对照组，但是各组织到达峰值的时间具有差异性 (P<0.01)，表明Ah胁迫下MHCⅠ基因在不同组织的表达时间存在差异。研究结果为进一步探究MHC分子在抗感染中的免疫功能提供了参考依据。

Abstract:

The aim of this study was to investigate the expression of MHCⅠ gene in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila (Ah), and to provide evidence for revealing the anti-infective immune response mechanism of amphibians. The experimental animal model of Aeromonas hydrophila infection was first constructed, and the pathological changes were observed by HE staining. The MHCⅠ gene α1+α2 peptide binding region of Rana dybowskii was cloned by RT-PCR and analyzed by bioinformatics. Real-time PCR was used to detect the transcription level of MHCⅠ in different tissues under Ah stress. After Ah infection, the skin, liver and muscle tissues showed signs of cell structure disappearance and texture disorder. The MHCⅠ gene α1+α2 peptide binding region fragment was 494 bp, encoding 164 amino acids, and homology with amphibians. Above 77%, the homology with mammals was as low as 14.96%, indicating that the α1+α2 region of MHC gene was less conserved among different species. The results of real-time PCR show that the liver, spleen and kidney of the experimental group were under Ah stress. The transcript levels of MHCⅠ gene in skin and muscle tissues were higher than those in the control group at 72 h, but the time to peak of each tissue was different (P<0.01), indicating that the response time of MHCⅠ gene in different tissues was different under Ah stress. This study provides a reference for further exploring the immune function of MHC molecules in anti-infection.