RNAI GENE SILENCING IN Lobesia botrana: DSRNA SYNTHESIS AND IN SILICO SPECIFICITY CONTROL

RESA JURIN, L.; LANZA VOLPE, M.; GOMEZ TALQUENCA, S.

San Juan

Congreso; XLI Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2023

Sociedad de Biología de Cuyo

RNAI GENE SILENCING IN Lobesia botrana: DSRNA SYNTHESIS AND IN SILICOSPECIFICITY CONTROLResa Jurin L, Lanza Volpe M, Gomez Talquenca SEEA Mendoza-INTA, Mendoza, Argentina. E-mail: resa.lucas@inta.gob.arLobesia botrana, the main grapevine pest, was introduced to Mendoza province in 2010. Local growers found themselvescompelled to implement phytosanitary measures, either through costly pheromone-based mating disruption or the applicationof insecticidal chemicals, with a substantial environmental impact and high risks to other organisms. The adoption of geneticsilencing via RNA interference (RNAi) for pest control presents a more cost-effective and environmentally friendly alternative.Therefore, researching and developing this technology is essential. Based on known transcripts of genes essential for survivalin other lepidopteran species, candidate sequences were chosen for RNA interference. The goals of this study were to confirmthe presence of these sequences within the pest while ensuring the absence of any transcript in other species that might besilenced off-target. Additionally, we aimed to synthesize double stranded RNA (dsRNA) molecules and confirm their structuralintegrity, before testing them on Lobesia botrana larvae. To achieve this, we conducted an in silico study by comparing thesesequences with a de novo assembled transcriptome of L. botrana in our laboratory, as well as with transcriptomes from differentspecies that might be impacted. Furthermore, primers were specifically designed for these sequences, and the presence of thetranscripts in RNA extracts from insect larvae was confirmed through PCR. Subsequently, we cloned the amplicons of eachtarget gene into the pGEM-T Easy vector. We designed a pair of primers with the T7 promoter sequence at their 5´ end toamplify the region containing the insert. Using these amplifications, flanked by the T7 promoter, we synthesized dsRNA invitro employing T7 RNA polymerase. Finally, we assessed the structural integrity of the dsRNA using RNase A, both in thepresence and absence of NaCl. The results demonstrate that the assessed sequences are present in the pest´s transcriptome butnot in those of other species. Consequently, these genes are promising candidates for evaluation as RNAi gene silencing targets.This entails supplementing larvae with newly synthesized dsRNA, evaluating the expression of these genes, and monitoringthe insect´s survival, development, and behavior.