中华绒螯蟹EsSox21b-like基因干扰载体的构建.docVIP

  中华绒螯蟹 EsSox21b-like 基因干扰载体的 构建# 刘志强，陈洁，邱高峰** 5 10 15 20 （上海海洋大学农业部淡水水产种质资源重点实验室） 摘要：Sox(SRY-related HMG-box)基因是一类重要的转录因子，在动物的性别分化与决定、 器官的发生等方面具有重要的调控作用。之前，我们研究组在中华绒螯蟹分离鉴定了一个只 在性腺特异性表达的 Sox 基因,命名为 EsSox21b-like，推测该基因可能与中华绒螯蟹的性腺 分化与发育有关。为了利用 RNA 干扰技术证实该基因的功能，本研究将 EsSox21b-like 基因 617617bp 的片段克隆至含有两个 T7 启动子的 L4440 载体上，构建了 EsSox21b-like-L4440 干扰载体，将该重组载体转化至 RNase缺陷型的 HT115 菌株中，经 IPTG 诱导菌株体内转 录 获 得 EsSox21b-like 正 义 和 反 义 单 链 RNA ， 经 过 变 性 、 退 火 形 成 大 量 双 链 RNA （EsSox21b-like-dsRNA），能满足以后应用 RNA 干扰试验研究 EsSox21b-like 基因的功能。 关键词：中华绒螯蟹；RNA 干扰；L4440；HT115；原核表达；dsRNA Construction of interference vector of EsSox21b-like gene of Chinese mitten crab ( Eriocheir sinensis) and preparation of dsRNA through prokaryotic expression LIU Zhiqiang, CHEN Jie, QIU Gaofeng (Key laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University) Abstract: Sox (SRY-related HMG-box) gene is a kind of important transcription factor, which functions in sex determination, organogenesis, etc. In our previous study , a novel gonad-specific 25 30 35 40 gene，named EsSox21b-like，was cloned from Chinese mitten crab (Eriocher sinensis). The gonad-specific expression of this novel gene suggested that it might play an important role in the development of gonad. To determine the exact role of EsSox21b-like gene using RNA interference, in this study we successfully constructed a EsSox21b-like-L4440 interference vector for production of large amount of dsRNA. The target sequence of EsSox21b-like gene was inserted into the L4440 vector containing two T7 promoters.. Then the recombinant plasmid was transformed into HT115 bacteria, a RNaseⅢ deficient strain. A large amount of EsSox21b-like sense and antisense RNAs was simultaneously transcripted in vivo when induced by IPTG. After denatured and annealed, EsSox21b-like double strand RNAs (dsRNAs) were produced, and the yield of EsSox21b-like-dsRNA was enough for future RNA interference experiment on the role of EsSo