Triazophos Up-regulated Gene Expression And Molecular Characterization Of Two Acetylcholinesterase Genes For Nilaparvata Lugens

The brown planthopper (BPH) Nilaparvata lugens (Sta1)(Hemiptera: Delphacidae) is one of the most serious insect pests of rice in Asia. Currently, chemical control of N. lugens primarily relies on the application of various insecticides, which gives rise to planthopper resurgence and insecticide resistance. N. lugens is the type of insect pest whose recurrence is induced by pesticides. Possible reasons include the destruction of the natural enemy community and the stimulation of pest reproductivity by sub-lethal dosages of some pesticides. However, the mechanisms of N. lugens resurgence and resistence due to insecticide stimulation are poorly understood. In this dissertation,we mainly studied the genes which are closely related to the mechanisms of N. lugens resurgence and resistance. Firstly, after triazophos treatment, the gene expression variation in female N. lugens nymphs was detected. Secondly, using reverse transcription-PCR and RACE method, the two acetylcholinesterase genes and their characterized were carried out. The expression levels of the two types of ace at different development stages and tissues were detected by RT-PCR and their related functions were indicated through the RNA interference experiment. The primary results are as follows:1. Triazophos up-regulated gene expression in the female brown planthopperWe investigated an organophosphorous insecticide, triazophos, and its ability to induce gene expression variation in female N. lugens nymphs just before emergence. By using the suppression subtractive hybridization method, a triazophos-induced cDNA library was constructed. In total,402differentially expressed cDNA cloneswere obtained. Real-time qPCR analysis confirmed that triazophos up-regulated the expression of six candidate genes at the transcript level in nymphs on day3of the5th instar. These genes encode N. lugens vitellogenin, bystin, multidrug resistance protein (MRP), purine nucleoside phosphorylase (PNP), pyrroline-5-carboxylate reductase (P5CR) and carboxylesterase. Our results imply that the up-regulation of these genes may be involved in the induction of N. lugens female reproduction or resistance to insecticides.2. Molecular cloning and characterization of two Nlaces genesIn this study, full-length cDNA sequences of two AChE genes (Nlace1and Nlace2) were sequenced from the brown plant-hopper (BPH) Nilaparvata lugens, the most destructive insect pest of rice crops. Nlace1cDNA is2842nucleotides long and contains an ORF potentially encoding a790amino acid peptide. Nlace2cDNA is2852bp in length and contains an ORF that potentially encodes a672amino acid peptide. Nlace1has an identity of40%with Nlace2at the amino acid sequence level. Phylogenetic analysis of59AChEs from32animal species available from GenBank showed that Nlace1is most closely related to AChE1s from Blattella germanica and Nephotettix cincticeps, while Nlace2is most closely related to AChE2from N. cincticeps. Quantitative RT-PCR analysis showed that Nlace1is expressed at a much higher level than Nlace2in all developmentalstages and tissues, demonstrating that Nllacel may be the dominant AChE form of the two enzymes. This result will help reveal the resistance mechanism of N. lugens to organophosphorous and carbamate insecticides and promote development of more selective insecticides targeting the main NlAChE1.3. RNAi analysis of two types AChE in Nilaparvata lugensTo obtain a direct measurement of differential contributions of AChE genes to AChE activity in N. lugens, we used dsRNA-mediated specific depletion of either Nlace1or Nlace2transcripts. Administration of dsRNA for Nlace1caused about more than70%reduction of the corresponding transcript on day3after injection without affecting that of Nlace2. The level of Nlace2transcript was decreased up to70%compared to the control3days after injection of dsNlace2. But no difference in N.lugens survival was observed among the RNAi groups and control, despite the fact that AChE expression level were significantly reduced. To assess whether reduction in AChE expression level by RNAi lead to increased sensitivity to organophosphates, dsNlace-injected insects were treated with chlorpyrifos by topical application at4days after injection. Bioassays on dsRNA-injected N. lugens indicated that partial reduction of Nlace1led to increased sensitivity to chlorpyrifos whereas Nlace2knockdown had a slight effect on mortality, suggesting that AChEl is the primary target of organophosphate insecticide in the female N. lugens and its quantity is related to chlorpyrifos sensitivity. In addition, we constructed eukaryotic expression vector of recombinant bacmid to express Nlaces in vitro. But there is no obvious expressed product detected in the experiment.