Biotechnology Bulletin ›› 2014, Vol. 0 ›› Issue (6): 134-138.

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

Liu Zhiqiang, Chen Jie, Qiu Gaofeng   

  1. Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306)
  • Received:2013-11-06 Online:2014-06-25 Published:2014-06-25

Abstract: 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 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 EsSox21b-like gene.

Key words: Eriocheir sinensis, RNA interference, L4440, HT115, Prokaryotic expression, dsRNA