| The brown planthopper (Nilaparvata lugens(Stal), BPH) is one of the major pests of rice in our Asia, it causes great yield loss of rice. It can migrate for long distance. The occurrence areas of BPH in China have been expanding in recent years, because of global climate anomalies, the changing of rice varieties and ecosystem, and the application of chemical fertilizers, pesticides etc.To clarify inherent mechanism of disasters caused by BPH, it is necessary to study the molecular mechanism of growth and development of BPH, by molecular biological technology. RNA interference (RNAi) is a powerful molecular tool, and has been widely used in functional genomics sdudies of insects.21E01 is a full-length cDNA clone from a normalized cDNA library of BPH, encoding V-ATPase E of BPH. The V-ATPase E likes membrane transporter binding protein, locates gastrointestinal epithelial cells of insect, it is the energy resource of plasma membrane alkaline and amino acids absorption in several insects. Three double-stranded RNAs (dsRNA) (ds1-21E01、ds2-21E01 and ds3-21E01) corresponding to the different sites of 21E01 were synthesized using gene specific primers in this study, and delivered into 3rd instar larve of BPH by artificial feeding, and the effects of three different dsRNA on target gene expression were analyzed. The results were as follows:1. Pure artificial diets of BPH have been made up successfully according to previous methods. The results of artificial feeding indicated that the survival rate of BPH was decreasing with time, however, the cumulative survival rate of BPH was more than 90%. This provided a good foundation for establishment of RNA interference technique by dsRNA feeding.2. The results showed that gene silencing could be induced by all of the 3 dsRNAs at 50 ng/μl in N. lugens. Time-dynamics course analysis of the gene silencing indicated that a significant suppression of target gene induced by ds2-21E01 and ds3-21E01 was observed as early as for two days, while that triggered by ds1-21E01 occurred as late as 10d of ingestion. The maximum reduction of V-ATPase-E mRNA occurred at 10d of exposure to dsRNA, ingestion of ds1-21E01, ds2-21E01, ds3-21E01 decreased gene expression by 41%,55% and 48% respectively.3. The mortality of BPH after dsRNA ingestion for two days, six days and ten days later was investigated in this study. The results showed that there was no significant difference in mortality among BPHs feeding on ds1-21E01, ds2-21E01, ds3-21E01 and ds-GFP, that is to say, the ds1-21E01、ds2-21E01 and ds3-21E01 have no lethal effect on BPH.4. When BPH nymphs fed with ds1-21E01、ds2-21E01 and ds3-21E01 emerged, they were coupled and maintained with rice seeding at tillering stage, the offsprings of them were collected. The expression level of 21E01 in offsprings of BPH treated with ds1-21E01 was significantly higher than that on ds-GFP. While the expression level of 21E01 in offsprings of BPH treated with ds2-21E01 and ds3-21E01 was not significantly different from that on ds-GFP. This indicated that under conditions of this study, there was no systemic RNA interference effects on BPH offsprings.An efficient and convenient RNA interference technique had been established this study, and this was the first research of RNA interference technique in BPH. This technique will provide powerful tools for functional genomics of BPH, and provide a new issues for BPH control by transgenic plants expressing dsRNA. |
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