Bacterial Expression Of DsRNA And Host Expression Of Hairpin DsRNA-mediated RNA Interference On Mythimna Separata Chitinase Gene

Mythimna separata(Corn armyworm)belongs to Lepidoptera Noctuidae and is one of the most important agricultural pests,causing severe crop losses by its outbreaks.RNAi(RNA interference)is a post transcriptional gene silencing mechanism mediated by endogenous or exogenous dsRNA(double-stranded RNA).In addition to its successful use in model insect D.melanogaster,RNAi has also been widely applied in non-model insects.Recent studies showed that silencing sequences with lethal effects on target insects could be easily found by the RNA interference technology,when combined with the second generation sequencing technology,as will accelerate the use of RNAi technology to control the crop pests.In this study,chitinase genes of corn armyworm were selected as the candidate genes,and interference sequence against chitinasel and chitinase2 genes were designed and synthesized.Two experimental procedures were designed to achieve the RNA interference effects.Firstly,bacterially expressed dsRNA using the L4440 vector and HT115 strain was applied to silence the expression of target genes.Interfering sequences against the target genes were cloned into the L4440 vector to produce sequence specific dsRNA,and recombinant vectors were transformed into Escherichia coli strain HT115(DE3)which is defective in dsRNA degradation activity.After IPTG induction,the dsRNA was purified and mixed with artificial diet and were fed to Mythimna separata.We showed that oral delivery of bacterially expressed double-stranded RNA(dsRNA)would lead to RNAi effects in the recipient insect as evidenced by the following experiments.Quantitative real-time PCR results showed that expression level of target Chi1 and Chi2 genes of Mythimna separata were down-regulated after oral delivery of engineered bacteria expressing the corresponding dsRNA after seven days' feeding.At the same time,the specific interference of chitinasel and chitinase2 genes also caused a corn armyworm weight loss and increase in mortality.Second,transgenic plants expressing hairpin dsRNA interference sequence to resistant against corn armyworm was developed.We constructed a dsRNA plant binary expression vector containing the target gene of hairpin using Gateway cloning technique.The results showed that both the entry vector and the plant binary expression vector were successfully constructed.The recombinant plant binary expression vectors were introduced into recipient rice to obtain transgenic rice.After kanamycin antibiotic screening and PCR detection,22 lines chi1 gene and 13 lines for chi2 gene were obtained.