黑龙江林蛙瘦素受体基因克隆及其在感染中的表达分析  

Cloning and Expression Analysis of LEPR Gene in Rana amurensis Exposed to Infection

作　　者：许冬梅 郭晶晶 任慧敏 赵雪滢 刘玉芬[1] 刘鹏[1] 赵文阁[1] XU Dong-Mei;GUO Jing-Jing;REN Hui-Min;ZHAO Xue-Ying;LIU Yu-Fen;LIU Peng;ZHAO Wen-Ge(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,哈尔滨150025

出　　处：《中国生物化学与分子生物学报》2023年第2期244-258,共15页Chinese Journal of Biochemistry and Molecular Biology

基　　金：黑龙江省自然科学基金联合引导项目(No.LH2021C053);黑龙江省大学生创新创业训练计划(No,202010231020)资助。

摘　　要：黑龙江林蛙(Rana amurensis)在东北地区森林生态系统保护中发挥重要作用,同时又具有很高经济价值。瘦素受体(leptin receptor,LEPR)属于I类细胞因子受体超家族成员,是调节能量代谢、调控免疫应答的关键因子。为研究LEPR基因在嗜水气单胞菌(Aeromonas hydrophila,Ah)感染中的作用,利用RT-PCR方法克隆黑龙江林蛙LEPR完整编码区,并对序列进行生物信息学分析;构建Ah感染的黑龙江林蛙炎症模型,通过qRT-PCR、免疫组织化学染色和Western印迹等方法,对LEPR在生理状态下及感染状态下不同组织的表达进行动态检测。结果显示,LEPR基因读码框含3 408bp,共编码1 135个氨基酸,其在黑龙江林蛙多个组织中均有表达,心血管表达量最低。在Ah感染下心、肝、脾、肾、皮肤、肌肉和胃LEPR mRNA均在72 h达到表达量峰值(P<0.05),肺组织在8 h达到峰值(P<0.05)。免疫组织化学和Western印迹显示,肝、脾、皮肤和胃之间LEPR表达量差异不显著(P>0.05);脾和皮肤在不同感染时间表达量从高到低为:72 h>对照>36 h,胃在36 h表达量最低(P<0.05)。综上所述,本研究成功克隆了LEPR基因,推断该基因参与了机体的感染过程,为进一步探究LEPR在两栖类的生物学功能奠定基础。Rana amurensis plays an important role in the protection of forest ecosystem in Northeast Chi-na and has high economic value.Leptin receptor(LEPR)is a member of class I cytokine receptor super-family,and is a key factor regulating energy metabolism and immune response.In order to study the role of LEPR gene in the infection of Aeromonas hydrophila,the complete coding region of LEPR of Rana amurensis was cloned by RT-PCR,and the sequence was analyzed by bioinformatics;The inflammatory model of Rana amurensis infected with Ah was constructed,and the LEPR gene in different tissues at dif-ferent time points after infection was detected by qRT-PCR.The expression levels of LEPR protein in liv-er,spleen,skin and stomach tissues were analyzed by immunohistochemistry and Western blot.The re-sults showed that the reading frame of LEPR gene contained 3408 bp,encoding a total of 1135 amino acids,and it was expressed in many tissues of healthy Rana amurensis,with the lowest expression in heart.Under Ah infection,the expression of LEPR mRNA in heart,liver,spleen,kidney,skin,muscle and stomach reached the peak at 72 h(P<0.05),and lung tissue reached the peak at 8 h(P<0.05).Immunohistochemical results and Western blot results showed that there was no significant difference in LEPR expression between liver,spleen,skin and stomach(P>0.05);The expression of LEPR in spleen and skin at different infection times ranged from high to low:72 h>control>36 h,the lowest ex-pression in stomach at 36 h(P<0.05).In conclusion,the LEPR gene was successfully cloned in this study,and it is inferred that this gene is involved in the body’s infection process,lays a foundation for further exploring the biological function of LEPR in amphibians.

关 键 词：黑龙江林蛙 瘦素受体 嗜水气单胞菌 炎症反应 

分 类 号：Q7[生物学—分子生物学]