Production of functional double-stranded RNA using a prokaryotic expression system in Escherichia coli

Abstract

RNA interference (RNAi) is a nucleic acid metabolism system utilized for the post-translational regulation of endogenous genes or for defense against exogenous RNA or transposable elements. Double-stranded RNA (dsRNA)-mediated RNAi shows broad application prospects to improve existing plant traits and combat invading pathogens or pests. To improve dsRNA transcriptional efficiency using a prokaryotic expression system, Trxz gene, an essential gene for the early development of chloroplasts in Arabidopsis thaliana, was chosen for a functional study. Two types of recombinant expression vectors, pDP-Trxz and phP-Trxz-N/L, were constructed to generate dsTrxz, the dsRNA which specifically induces Trxz gene silencing. Gel electrophoresis tests showed that phP vectors performed better and produced more dsRNA than the pDP vector under the same conditions. Purification of dsTrxz by enzymatic digestion indicated that highly purified dsRNA can be obtained through the use of DNase enzymatic hydrolysis assay. To confirm the knockdown effect of the dsRNA, a root immersion assay was performed, and we found that the root immersion culture could continue to affect the growth and development of A. thaliana. This included inhibiting the development of new leaves, causing weak plant development, leaf whitening, and other symptoms. This indicated that in vitro expressed dsRNA can be absorbed through Arabidopsis roots and can continue to trigger Trxz gene silencing. To delay dsRNA degradation and extend the effectiveness of RNAi, nanomaterial layered double hydroxide (LDH)-mediated BioClay was performed. We found that LDH-mediated BioClay alleviates the degree of dsRNA degradation, which provides a new idea for the storage and transportation of dsRNA.