利用细菌表达dsRNA介导黄粉虫抗冻蛋白基因的RNA干扰

摘要

RNA 干扰（RNA interference，RNAi）是研究基因功能的一种重要工具。为了利用RNAi技术对黄粉虫抗冻蛋白（Antifreeze protein，AFP）基因的非抗冻功能进行验证，将黄粉虫抗冻蛋白基因Tmafp433的相应干扰片段构建至L4440干扰载体并转化大肠杆菌HT115（DE3）菌株，利用IPTG诱导表达特异的afp基因相应dsRNA，纯化后注射黄粉虫幼虫，通过实时荧光定量PCR检测afp基因在mRNA水平的变化。结果显示含有L4440-Tmafp重组质粒的HT115菌株可以表达干扰afp基因的dsRNA，命名为Tmafp-dsRNA。用Tmafp-dsRNA注射黄粉虫24 h后，Tmafps的表达受到显著抑制，相比对照下降了60.8%。本研究表明通过注射dsRNA可有效抑制黄粉虫afp基因的表达。%In order to further study the non-freezing function of antifreeze protein(AFP)genes in Tenebrio molitor using RNAi technology, the RNAi fragment of Tmafp433 from T. molitor was inserted into L4440 dsRNA expression vector, and transformed into E. coli HT115(DE3). The dsRNA corresponding to Tmafps, designated as Tmafp-dsRNA, was expressed by IPTG induction, and injected into the larvae after purification. The mRNA level of Tmafps was detected using real-time quantitative PCR. The results showed that the E. coli HT115 (DE3)containing L4440-Tmafp recombinant plasmid can express Tmafp-dsRNA. After 24 h of injection, the expression of Tmafps in T. molitor was significantly decreased to 60.8%of the control, suggesting that the expression of Tmafps was inhibited by injecting the bacterially expressed Tmafp-dsRNA. The present results laid foundation for further research in afp gene function.