RNAi Effects Toward Chitinase Genes In Mythmina Separata By Oral Delivery Of Double-Stranded Rnas And Recombinant Tobacco Mosaic Virus

RNAi (RNA interference) is a technology used for silencing of target genes via sequence-specific manner. There are already many examples and even practical implementation of RNAi-based technologies for development of anti-pathogenic crops. In 2007, researchers from Monsanto and CAS reported the development of transgenic corn and cotton resistant to insect herbivore using RNAi technology, providing a species-specific and environmentally sound anti-pest strategy. Mythimna separata belongs to noctuidae family of lepidoptera and is harmful to crops of economic importance. Recently, outbreaks of Mythimna separata severely threatens corn production in Northern China, calling for new control approaches.We propose here to develop Mythimna separata-resistant plants using RNAi technology. Our primary hypothesis is that if we can identify and deliver to Mythimna separata interfering RNAs that down-regulate expression of Mythimna separata genes such as Chitinase genes which are essential for their growth and development, they will result in a debilitating phenotype. To do this, we designed and carried out the following experiments. First, in vitro synthesized dsRNA corresponding for interfering sequences against the target genes chitinase 1 and chitinase2 were mixed with artificial feed and were fed to Mythimna separata. Second, the interfering sequences against the candidate genes were cloned into plant viral vectors TMV (pJL36). Plant hosts such as N. Benthamiana was infected with the recombinant virus carrying insect sequences, and would produce the dsRNA and siRNA for the insert sequences during the process of RNA virus replication. The infected plants was fed to mythimna separata, as we expect results in degradation of target mRNA in the recipient insects.The RNAi effects toward endogenous complementary target genes then were evaluated by the following methods. Quantitative real-time PCR results showed that expression level of target chitinasel and chitinase2 genes in gut tissues of mythimna searata were down-regulated after oral delivery of both in vitro synthesized dsRNA and recombinant virus carrying interfering sequences. Furthermore, sequence-specific siRNA was detected in recipient insects, supporting the existence of siRNA-mediated silencing effects in mythimna separata. In addition, silencing of chitinasel and chitinase2 genes led to phenotypic effects such as reduced body weight and increased mortality.This reaserch provides an experimental foundation for using RNAi to control mythimna separata. The effective sequences, especially those caused phenotypic effects, will be cloned into plant binary vector. The recombinant binary vectors will be transformed into plant host to express hairpin dsRNA for the insert sequences, and the transgenic plants will be evaluated for the RNAi efficacy toward the feeding herbivore insects in our future study.