草鱼BAC文库中TLR3基因的筛选及表达特征的研究  

Screening and expression analyses of TLR3 from grass carp bacterial artificial chromosome (BAC) library

作　　者：杨春荣[1] 苏建国[1] 张荣芳[1] 董捷[1] 汪亚平[2] 张成勋[2] 

机构地区：[1]西北农林科技大学陕西省农业分子生物学重点实验室,陕西杨凌712100 [2]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,湖北武汉430072

出　　处：《西北农林科技大学学报（自然科学版）》2010年第6期83-87,共5页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(30871917);教育部新世纪优秀人才支持计划项目(NCET-08-0466)

摘　　要：【目的】从草鱼的BAC文库中筛选TLR3基因的阳性克隆,研究该基因在草鱼体内的表达规律,为进一步研究奠定基础。【方法】用同位素P32标记的探针,对53 216个BAC克隆以双份平行形式制备的DNA尼龙膜进行杂交筛选,用半定量方法检测TLR3在草鱼不同组织(鳃、心脏、肠、肝胰脏、肌肉、皮肤、脾脏、头肾和中肾)中的表达模式,用实时荧光定量RT-PCR研究草鱼感染呼肠孤病毒后不同时间TLR3的表达规律。【结果】获得了2个TLR3基因的阳性BAC克隆,该基因在被检测的9个组织中都有表达,其中在脾脏、皮肤中的表达量较高。在感染呼肠孤病毒后24 h,草鱼肝胰脏中TLR3的相对表达量显著升高(P<0.05);感染后48 h,其相对表达量恢复到正常水平(P>0.05)。【结论】成功筛选到草鱼TLR3的BAC克隆,该基因在草鱼体内不同组织中有广泛表达,并且参与了草鱼抗呼肠孤病毒的过程。【Objective】 The study screened TLR3（Toll-like receptor 3） gene from grass carp BAC（bacterial artificial chromosome） library and explored its expression profile in vivo.【Method】 Probe labeled by P32 was used to screen DNA nylon membrane of BAC library of 53 216 clonies duplicately.The TLR3 expression pattern was examined in different tissues（gill,heart,intestine,hepatopancreas,muscle,skin,spleen,head kidney and trunk kidney） by semi-quantitative RT-PCR.The transcripts were investigated by real time fluorescent quantitative RT-PCR after GCRV（grass carp reovirus）infection.【Result】 Two positive TLR3 clonies were obtained.TLR3 expresses in the nine tested tissues with high expression in spleen and skin tissues.The mRNA expression in hepatopancreas was significantly up-regulated at 24 h post-injection（P0.05） and came to the control level at 48 h post-injection（P0.05）.【Conclusion】 Two confirmed BAC clonies of grass carp TLR3 have been obtained.The TLR3 expression is widespread in the different tissues.The TLR3 gene participates in the process of anit-GCRV.

关 键 词：草鱼 BAC文库 TLR3基因 基因表达 

分 类 号：S965.112[农业科学—水产养殖] Q786[农业科学—水产科学]