Construction of Henosepilachna Vigintioctomaculata cDNA Library and Identification of dsRNA Injection–Induced RNAi Effect

Henosepilachna vigintioctomaculata is a pest that damages plants of the Solanaceae family. To control of H. vigintioctomaculata through RNAi, which has received much attention in recent years, we investigated the effect of RNAi by injecting dsRNAs and constructing a cDNA library for selecting the genes to be targeted by RNAi. The N20 primer was used to synthesize cDNA of different sizes by removing the poly A. After amplification, PCR products of sizes varying between 200 and 600 bp were obtained. For the efficient cloning of the different genes, the products were cloned into a pDONR 207 vector using the Gateway system for constructing the cDNA library. The cDNA library thus constructed had a titer of 3.15 × 10⁵ cfu/ml. The genes from the cDNA library were cloned into a LITMUS 28i vector containing the T7 promoter for synthesizing dsRNA. Electro–transformation was performed in the E. coli cells, and 48 colonies were randomly selected to confirm the size and duplication of the insert. The insert sizes varied between 100 and 500 bp and the different genes were cloned without duplicating the sequences. The results showed that 23 genes were those of insects, including those of the order Coleoptera, while the rest were genes from non–insect species. Using the T7 promoter of the LITMUS 28i vector, the genes with identified sequence and gene information were used for synthesizing 200–600 bp dsRNAs suitable for RNAi. Most of the larvae injected with Hv1, Hv4, and Hv7 genes turned black and died within 7–10 days after injection.