RNAi of a chymotrypsin gene of Helicoverpa armigera by using of dsRNA expressing recombinant bacteria
عنوان دوره: دومین کنگره بین المللی حشره شناسی ایران
نویسندگان
چکیده
RNAi (RNA interference) is a powerful tool for silencing of target genes through sequence-specific manner. Helicoverpa armigera belongs to Noctuidae family of Lepidoptera and is serious threat to crops of economic importance. One of H. armigera chymotrypsin gene (HaCHY) basis of our previous results, was chosen as the target gene as they were expressed predominantly in the gut tissue and were reported to be ideal silencing targets in insect species. Target sequence of the gene was cloned into the L4440 vector to produce sequence specific dsRNAs (double-stranded RNAs). Recombinant L4440 vectors were transformed into Escherichia coli strain HT115 (DE3) which is incapable in dsRNA degradation activity, so shielding the dsRNA from degradation by cellular systems like some RNases. The artificial diet was mixed with the bacteria (108 spores/ml) and were delivered to H. armigera. Our results showed that oral delivery of bacterially expressed dsRNA let to RNAi effects in the treated insect larvae. Quantitative real-time PCR results showed that expression level of target HaCHY gene in gut tissue of H. armigera was down-regulated post feeding of engineered bacteria expressing the corresponding dsRNA. Pretreatment with an ultra-sonication and heating to disrupt bacterial cell wall/membrane significantly increased the insecticidal activity of the transformed bacteria. Furthermore, knockdown of HaCHY gene resulted in increased mortality and reduced body weight of the feeding larvae. Our research provides both an experimental foundation for using RNAi technology to control H. armigera and also a useful research tool for loss-of-function study of important developmental and regulatory genes in this insect species.
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