中华绒螯蟹EsSox21b-like基因干扰载体的构建及原核表达制备dsRNA  

Construction of Interference Vector of EsSox21b-like Gene from Chinese Mitten Crab (Eriocheir sinensis) and Preparation of dsRNA by Prokaryotic Expression

作　　者：刘志强[1] 陈洁[1] 邱高峰[1] 

机构地区：[1]上海海洋大学农业部淡水水产种质资源重点实验室,上海201306

出　　处：《生物技术通报》2014年第6期134-138,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31272655);国家科技支撑计划项目(2012BAD26B04);上海市科委基础重点科研项目(11JC1404600);上海高校水产学一流学科建设项目(B5005120001)

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。旨在利用RNA干扰技术证实该基因的功能,将EsSox21b-like基因617 bp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RNaseⅢ缺陷型的HT115菌株中,经IPTG诱导菌株体内转录,获得了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干扰试验对于EsSox21b-like基因功能的研究。Sox（SRY-related HMG-box）gene is a kind of important transcription factor, which functions in sex determination, organogenesis, etc. 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, a EsSox21b-like-L4440 interference vector was successfully constructed 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 RNaseⅢdeficient 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 EsSox21b-like gene.

关 键 词：中华绒螯蟹 RNA干扰 L4440 HT115 原核表达 DSRNA 

分 类 号：Q78[生物学—分子生物学]