Comparative Analysis of In Vitro vs. In Vivo dsRNA Production for CHS Silencing and Downstream Flavonoid Pathway Suppression in Arabidopsis thaliana

Exogenously induced RNA interference (exoRNAi) is a powerful biotechnology tool for precise gene regulation. The plant chalcone synthase (CHS) gene serves as a valuable model for molecular biology due to its central role in flavonoid biosynthesis. However, there are currently very few studies addressing the advantages and disadvantages of in vitro (enzymatic) or in vivo (bacterial) methods for producing double-stranded RNA (dsRNA) for exogenous application. This study aims to optimize and compare the two methods for producing dsRNAs targeting the Arabidopsis thaliana CHS gene: enzymatic synthesis in vitro using a commercial kit and bacterial synthesis in vivo using an engineered E. coli HT115 (DE3) system. Bacterial synthesis conditions were optimized with respect to IPTG concentration and cultivation time, and the resulting dsRNA preparations were purified and quality-controlled. Their biological activities were assessed by treating A. thaliana plants and analyzing the effects on AtCHS gene expression and flavonoid production using qRT-PCR and HPLC-MS. The results demonstrated that purified AtCHS-dsRNA from both methods effectively suppressed AtCHS expression and downstream flavonoid biosynthetic gene expression, leading to significant reductions in anthocyanins and flavanols. This study confirmed the efficacy of exogenous dsRNAs in regulating plant metabolic pathways and provided a comparative analysis of dsRNA synthesis methods, highlighting their benefits and limitations for practical applications in plant biology and protection.