杂色鲍原代细胞培养方法的优化及其在基因功能研究中的应用  

Optimizing Primary Cell Culture Method of Haliotis diversicolor and Its Effect for Gene Function Study

作　　者：李敏[1,2] 张丽莉[1,2] 王国栋[1,2] 张龙辉[1,2] 王艺磊[1,2] 

机构地区：[1]集美大学水产学院农业部东海海水健康养殖重点实验室,福建厦门361021 [2]集美大学水产生物技术研究所,福建厦门361021

出　　处：《安徽农业科学》2015年第15期146-150,共5页Journal of Anhui Agricultural Sciences

基　　金：国家自然科学基金项目(41006105;41176152);福建省科技计划重点项目(2011N0022)

摘　　要：优化了杂色鲍原代细胞的培养条件,并利用原代细胞进行了RNAi和外源基因表达。结果表明,较高的渗透压对血淋巴细胞培养起重要作用,淋巴细胞分离液分离血淋巴细胞更利于其生长。原代细胞培养至第5天,在细胞培养液中添加50μg My D88 dsRNA,能够显著降低My D88 mRNA的表达。p EGFP-N1转染培养5 d的鳃细胞,5 d后可以检测到绿色荧光。该研究表明可以成功培养杂色鲍血和鳃原代细胞,并且利用原代细胞可以进行功能基因表达量调低或调研究,为功能基因注释提供了便利条件。The condition of primary cell culture was optimized in Haliotis diversicolor and these primary cells of hemocyte and gill were used in RNA interference（RNAi） and exogenous gene expression.Results suggested that hemocyte survived better in higher osmolarity of media,the way of using lymphocyte separation medium to culture hemocyte was beneficial for cell growth.My D88 dsRNA were added to cell culture medium of hemocyte and gill after 5 days of culture which reduced the expression of My D88.The plasmid p EGFP-N1 was transfected into gill after5 days of culture and the green fluorescence was detected 5 days later.Above all,the primary cell can survived in vitro and they are a proper tool for gene function analysis by regulating gene expression up or down,which provide convenient way for functional annotation.

关 键 词：杂色鲍 原代细胞培养 RNAI 转染 

分 类 号：S917[农业科学—水产科学]