| The beet armyworm, Spodoptera exigua (Hubner), is one of the most serious agricultural pests of vegetable and flower crops in many agricultural areas all over the world. Unfortunately, the resistance caused by long-term use of insecticides is gradually enhanced, so efficient control technologies of the beet armyworm are needed to substitute traditional chemical control. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step is to find suitable target genes, based on sequence similarities with known target genes in the corn rootworms and the importance of genes in cellular process, we chose nine candidate genes to study their potential application in RNAi-mediated pest control. The research provides the theory basis for safety and effective use of RNAi-mediated pest control technique. The main results are summarized as follows:1. Gene cloning of the candidate genes in the beet armywormWe used rapid amplification of cDNA ends (RACE) to amplify the full-length transcripts of the selected candidate genes. We successfully amplified the5’-UTR sequences of six genes (arfl, arf2, tubulin2. chitinase7, PGCP, helicase), but only obtained the complete3’-UTR sequence of arf2. We assembled the5’UTR, fragments from transcriptome and3’UTR into a single transcript. Only the full-length transcript of arf2gene was successfully obtained. Five genes (arfl, arf2, tubulin2, chitinase7, helicase) had an intact CDS. These sequences shared high similarities with their orthologous in other insects. All sequences were submitted to the GenBank database.2. Knock down of candidate genes by siRNAs injectionWe designed siRNAs based on the sequences confirmed by end-to-end PCRs, and injected2μl siRNA (2μg/μl) into the4th instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (-20-94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulinl, arf2, tubulin2and arfl caused a significantly high level of mortality compared to the negative control (P<0.05). About80%of the survived insects in the siRNA-treated group of five genes (PGCP, chitinasel, tubulinl, tubulin2and helicase) showed retarded development. In chitinasel-siRNA and chitinase7-siRNA administered groups,12.5%survivors exhibited "half-ecdysis". In arfl-siRNA and arf2-siRNA groups, the body color of15%became black48h after injections.3. The off target effects in RNAi of the beet army wormWe choose chitinasel and arf2genes to do the off target effects according to the RNAi in the beet armyworm. After injecting siRNAs, we detected the mRNA levels of the non-target genes by DGE (Digital Gene Expression Profiling) and qRT-PCR respectively. The non-target genes are the genes which different matched with the siRNA of chitinase1and the siRNA of arf2gene, which are homologous with chitinase1and arf2gene and which are in the metabolism pathway of chitinasel and arf2. This result of DGE and qRT-PCR showed that the mRNA levels of some of these non-target genes are up and down at different degree, it indicated that RNAi may cause off target effects for the frist and second types non-target genes, but the third types may caused by a chain reation of the metabolic pathway.4. The safety assessment of RNAiSilkworm (Bombyx mori) was taken as the research object, we injected2μl siRNA (2μg/μl) of the chitinasel and arf2genes into the3th instar larvae. We detected the mRNA levels of the non-target genes by qRT-PCR. The non-target genes are the genes which different matched with the siRNA of chitinasel and the siRNA of arf2gene and which are homologous with chitinase1and arf2gene. The result showed that the mRNA levels of some of these non-target genes are up or down at different degree, but we did not observe any retarded development, abnormal phenotypes and death in the experiment. |
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