东北林蛙LEPR基因的克隆及其在感染中的表达分析  

Cloning and Expression Analysis of Rana dybowskii LEPR Gene During Infection

作　　者：许冬梅 刘婷婷[1] 刘依铭 刘玉芬[1] 刘鹏[1] 赵文阁[1] XU Dongmei;LIU Tingting;LIU Yiming;LIU Yufen;LIU Peng;ZHAO Wenge(College of Life Science and Technology,Harbin Normal University,Harbin 150025,China)

机构地区：[1]哈尔滨师范大学生命科学与技术学院,黑龙江哈尔滨150025

出　　处：《华北农学报》2023年第3期227-238,共12页Acta Agriculturae Boreali-Sinica

基　　金：黑龙江省自然科学基金联合引导项目(LH2021C053)。

摘　　要：为了探究LEPR基因在两栖类的生物学功能,利用嗜水气单胞菌感染东北林蛙建立炎症模型,分析LEPR基因在细菌感染后的表达情况。首先利用RT-PCR技术克隆LEPR基因并进行生物信息学分析,再构建Ah感染的东北林蛙炎症模型;通过苏木精-伊红染色法(HE染色)观察感染后组织病理变化,并利用qRT-PCR技术分析生理状态和感染状态LEPR基因的组织表达规律差异,最后结合免疫组织化学染色对肾脏、皮肤和肌肉等组织中LEPR蛋白表达变化进行检测。结果显示,获得东北林蛙LEPR基因序列长度为3 604 bp,其开放读码框为3 405 bp,共编码1 134个氨基酸;亚细胞定位显示,LEPR蛋白质为具有一次跨膜结构域的膜蛋白;同源性分析证实东北林蛙与两栖类同源性在52.6%以上,表明LEPR的保守程度较低;基于qRT-PCR结果显示,LEPR mRNA在健康东北林蛙心脏、肝脏、脾脏、肺脏、肾脏、皮肤、肌肉和胃等8种组织中均有表达,在皮肤组织中的相对表达量显著高于其他组织;Ah感染后LEPR基因在不同组织中显著上调,但应答时间和水平有所差异;免疫组织化学结果表明,肾脏、皮肤和肌肉LEPR蛋白表达量变化趋势与qRT-PCR结果基本一致。皮肤组织中LEPR基因应答强烈,也说明其可能参与感染过程,为进一步扩展两栖类LEPR基因的免疫学功能研究奠定了基础。In order to explore the biological function of LEPR gene in amphibians,we infected Rana dybowskii with Aeromonas hydrophila(Ah)to establish an inflammatory model,and analyzed expression profile of LEPR gene after Ah infection.Firstly,the LEPR gene was cloned by RT-PCR technology and bioinformatics analysis was performed,and then the inflammation model of Rana dybowskii was constructed.The histopathological changes during infection were observed by hematoxylin eosin staining(HE staining),and the differences of tissue expression of LEPR gene in physiological and infectious states were analyzed by qRT-PCR.Finally,the changes of LEPR protein expression in kidney,skin and muscle tissues were verified by immunohistochemistry staining.The results showed that the length of LEPR gene sequence of Rana dybowskii was 3604 bp,and its open reading frame was 3405 bp,encoding 1134 amino acids.Subcellular localization showed that the LEPR protein was a membrane protein with a primary transmembrane domain.The homology analysis confirmed that the homology between Rana dybowskii and amphibians was more than 52.6%,indicating that LEPR was less conservative.Based on qRT-PCR results,LEPR mRNA was expressed in the heart,liver,spleen,lung,kidney,skin,muscle and stomach of the healthy Rana dybowskii,and its relative expression in skin tissue was significantly higher than that in other tissues.After Ah infection,the LEPR gene was significantly up-regulated in different tissues,but the response time and level were different.Immunohistochemical results showed that the change trend of LEPR protein expression in kidney,skin and muscle was basically consistent with that of qRT-PCR.The strong response of the LEPR gene in the skin tissue also indicates that it may participate in the infection process,which lays a foundation for further expanding the immunological function of the amphibian LEPR gene.

关 键 词：东北林蛙 嗜水气单胞菌 LEPR基因 组织表达 

分 类 号：Q956[生物学—动物学]