title: Second-generation sequencing supply an effective way to screen RNAi targets in large scale for potential application in pest insect control.
abstract: The key of RNAi approach success for potential insect pest control is mainly dependent on careful target selection and a convenient delivery system. We adopted second-generation sequencing technology to screen RNAi targets. Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) technologies were used to screen optimal RNAi targets from Ostrinia furnalalis. Total 14690 stage specific genes were obtained which can be considered as potential targets, and 47 were confirmed by qRT-PCR. Ten larval stage specific expression genes were selected for RNAi test. When 50 ng/µl dsRNAs of the genes DS10 and DS28 were directly sprayed on the newly hatched larvae which placed on the filter paper, the larval mortalities were around 40∼50%, while the dsRNAs of ten genes were sprayed on the larvae along with artificial diet, the mortalities reached 73% to 100% at 5 d after treatment. The qRT-PCR analysis verified the correlation between larval mortality and the down-regulation of the target gene expression. Topically applied fluorescent dsRNA confirmed that dsRNA did penetrate the body wall and circulate in the body cavity. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost less and an easy way to select the candidate target genes for RNAi. More importantly, it demonstrated that dsRNAs are able to penetrate the integument and cause larval developmental stunt and/or death in a lepidopteron insect. This finding largely broadens the target selection for RNAi from just gut-specific genes to the targets in whole insects and may lead to new strategies for designing RNAi-based technology against insect damage.
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, People's Republic of China.
authors.forename: Yubing
authors.initials: Y
authors.lastname: Wang
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Haichao
authors.initials: H
authors.lastname: Li
authors.forename: Xuexia
authors.initials: X
authors.lastname: Miao
mesh_terms: D000818:Animals; D001483:Base Sequence; D005456:Fluorescent Dyes / Q000378:metabolism; D018628:Gene Dosage / Q000235:genetics; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D007814:Larva / Q000235:genetics; D007915:Lepidoptera / Q000235:genetics / Q000254:growth & development; D008018:Life Cycle Stages / Q000235:genetics; D008969:Molecular Sequence Data; D010572:Pest Control, Biological / Q000379:methods*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D012330:RNA, Double-Stranded