BmICE-2 interference vector construction and its silencing effect in the BmN-SWU1 cells

摘要

Investigate the function of BmICE-2 gene in silkworm cells by constructing a BmICE-2 interference vector and analysing its interference effect. Online RNAi design software was used to predict the interference target sequence of Bmice-2. An optimized sequence was selected and inserted into the backbone of interference microRNA. The backbone of interference vector, microRNA-siBmICE-2, and a control vector, microRNA-siBmICE-2-control were designed. The synthesized gene segment was digested by Asc I and Sac II, and inserted into a PIZ/V5-dsRed vector. The interference vector PIZ/V5-BmICE-2-miRNA-dsRed and control vector PIZ/V5-BmICE-2-Control-miRNA-dsRed were designed and validated through digestion and sequencing. The interference and control vectors were then transfected into silkworm cells BmN-SWU1. qRT-PCR and Western blots were used to evaluate the effect of gene interference. Interference microRNA vector PIZ/V5-BmICE-2-miRNA-dsRed targeting BmICE-2 was successfully constructed and transiently expressed in silkworm cells. The interference vector significantly inhibited the expression of BmICE-2 at mRNA and protein levels. The constructed interfering vector significantly interfered with the expression of BmICE-2 in silkworm cells BmNSWU1.