Establishment of Bacillus thuringiensis based exogenous double-stranded RNA production platform

Abstract

RNA interference (RNAi) is a post-transcriptional gene regulation mechanism found in virtually all plants and animals including insects. RNAi has been considered as an alternative strategy to control agricultural pests whereby double-stranded RNA (dsRNA) triggers a potent and specific inhibition of its homologous mRNA. It also can be as potential therapeutic strategy for treating and preventing of diseases in beneficial insects for crops. Since small dsRNAs are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. In this study, to produce exogenous dsRNA through simple and cost-effective methods, there is a focus on developing entomopathogenic bacteria Bacillus thuringiensis. The first step was to find a target that confirming dsRNA triggered gene silencing phenomenon such as RNAi system in Sacbrood virus (SBV). It is a classified as the order Picornavirales, family Iflaviridae, genus Iflavirus with a positive single-stranded RNA genome, is one of the most fatal emerging honeybee virus that cause a serious threat to the Asian honeybee Apis cerana, and tends to cause bee colony and even the whole apiary collapse. In this research, dsRNAs targeting SBV structural protein were synthesized to control SBV. These dsVP1, dsVP3, and dsRdRp took RNAi effects in the SBV infected Apis cerana workers, as fed with these dsRNAs and confirmed that among the three dsRNAs, vp1 gene specific dsRNA (dsVP1) showed a powerful effect of reducing SBV replication. For the second step, it was necessary to use the characteristics of Bacillus thuringiensis. The gram-positive bacterium Bacillus thuringiensis has insecticidal proteins produced during the stationary/sporulation phase of growth with expression of their encoding genes driven by sporulation dependent promoters. To develop a dsRNA mass-production platform utilizing Bacillus thuringiensis, the pHT1K-SBV vp1 and pHT1K-EGPF plasmid vectors which transcribe sense and anti-sense target gene under the control of cyt1Aa sporulation-dependent promoter with STAB-SD sequence were constructed. According to the confirmation of target gene expression by reverse transcription PCR, relative transcription level by qPCR analysis, and dsRNA quantification by hybridization analysis, it suggested that a potential of Bacillus thuringiensis becoming a new platform in dsRNA production. Finally, to confirm if these dsRNAs play their role in RNAi system properly, the bioassay using SBV infected Apis cerana was conducted. Ingestion of these dsRNAs derived from transformed Bacillus thuringiensis to SBV infected Asian honeybee, Apis cerana showed powerful effect on SBV suppression. As a result, it was confirmed that the dsRNAs derived from B.thuringiensis play their role in RNAi system.