作　　者： ; ; ; ;

机构地区： 中国科学院南海海洋研究所

出　　处： 《中国水产科学》 2011年第2期379-391,共13页

摘　　要： 应用抑制性消减杂交技术(SSH)构建红笛鲷(Lutjanus sanguineus)头肾消减cDNA文库,筛选红笛鲷免疫相关基因的EST。以哈氏弧菌(Vibrio harveyi)灭活疫苗体内诱导红笛鲷为实验组,以注射无菌生理盐水的红笛鲷为驱动组,通过SSH技术构建红笛鲷头肾消减cDNA文库。利用PCR技术和斑点杂交对文库进行筛选,从2424个含插入片段的阳性克隆中筛选了680个克隆在上海生工进行了序列测定。使用BLASTx和BLASTn工具对获得的ESTs与GenBank数据库进行同源性比较并根据相似性序列的名称通过GO法对ESTs进行注释。结果获得了30个与红笛鲷免疫防御相关基因的EST,如组织相容性抗原复合物基因(MHCI和MHCⅡ)、免疫球蛋白基因(IgH和IgL)、热休克蛋白基因(HSP10、HSP70和HSP90)等。本研究构建了哈氏弧菌灭活疫苗免疫后与正常组织差异表达的消减cDNA文库,并获得一批与红笛鲷免疫防御相关的ESTs,旨在为探讨红笛鲷分子免疫防御机制、筛选参与免疫防御调控相关的功能基因,揭示红笛鲷免疫抗病机制、提高机体抗病力、实现遗传改良奠定基础。 A subtracted cDNA library of humphead snapper （Lutjanus sanguineus） was constructed by suppression subtractive hybridization technology （SSH） to screen immune-related EST.The cDNA library has been constructed by the mRNA of the test group and driven by SSH.Differential ESTs from the subtracted cDNA library have been identified by both PCR technology and dot blot hybridization.Six hundred and eighty positive clones were sequenced by Sangon Biological Engineering Technology Services Co.,Ltd.The homology of the sequences was analyzed by BLASTx tool and BLASTn tool in GenBank database.Functional distribution was performed based on the features of ESTs by gene ontology annotation （GO） and 30 immune-related ESTs of L.sanguineus,such as major histocompatibility complex gene （MHC Ⅰ and MHC Ⅱ）,immunoglobulin gene （IgH and IgL）,heat shock protein gene （HSP10,HSP70 and HSP90） and so on,were found.In this study,the subtracted cDNA library was successfully constructed by SSH and a number of immune-related ESTs of L.sanguineus were identified,which laid a foundation for the immune-related genes research of L.sanguineus.

关 键 词： 红笛鲷 抑制性消减杂交技术 文库 免疫基因

领　　域： [农业科学] [农业科学]