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Olfactory Genes Of Two Important Rice Insect Pests,Cnaphalocrocis Medinalis And Chilo Suppressalis

Posted on:2014-02-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:S LiuFull Text:PDF
GTID:1223330395493639Subject:Agricultural Entomology and Pest Control
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The rice leaffolder, Cnaphalocrocis medinalis (Guenee) and the rice stem borer, Chilo suppressalis (Walker) are notorious insect pests that cause considerable economic damage to cultivated rice crops each year in Asia. In this dissertation, key genes involved in olfactory signals processing of C. medinalis and Ch. suppressalis were studied, and the results were listed as follows:1) We isolated five full-length cDNA sequences encoding putative odorant-binding proteins (OBPs) from C. medinalis, named CmedGOBP1, CmedGOBP2, CmedPBPl, CmedOBP1and CmedOBP2. Real-time quantitative PCR (qPCR) was performed to investigate relative expression levels of these OBPs in different tissues and between male and female C. medinalis. The five OBPs were both antennae-specific genes with little trace were detected in other non-olfactory tissues, except CmedOBP1and CmedOBP2, which were detected in abdomen, wing and leg with considerable amount. DNA sequence encoding mature CmedGOBP2was ligated into pET-22b vector and transfered into Escherichia coli OrigamiB (DE3) cells, expression product was purified by ion-exchange chromatography and used in fluorescence competitive binding assay, using1-NPN as probe. In the binding assay, one pheromone constituent Z13-18:Ald, shows highest binding affinities to the CmedGOBP2, can displace1-NPN in a dose-dependent manner. In this process, CmedGOBP2may undergo a pH-mediated conformational change.2) Two full-length cDNA sequences encoding novel Orcos (CmedOrco and ChsupOrco) were cloned from C. medinalis and Ch. suppressalis, by using homologous clone strategy and RACE method. Bioinformatic prediction and cellular immunofluorescence indicated that CmedOrco and ChsupOrco are both seven-transmembrane receptors with intracellular N-termini and extracellular C-termini, which adopt a reverse membrane topology compared to the vertebrate G protein-coupled receptors (GPCRs). mRNA expression levels of the two Orcos are much higher in male and female antennae than those in non-olfactory tissues, and the ChsupOrco transcripts reach a peak level in adults compare to other life stages.3) Four candidate SNMP gene sequences were isolated from antennal cDNA of C. medinalis and Ch. suppressalis, named CmedSNMP1, CmedSNMP2, ChsupSNMP1and ChsupSNMP2. Prediction with the TMHMM and TMpred program indicated that the amino acid sequence of these SNMPs encoded putative α-helical2-pass transmembrane domain which is close to N-and C-terminus. We calculated the ratios of non-synonymous (dN) and synonymous (dS) changes of SNMP-coding nucleotide sequences in lepidopteran insects (including CmedSNMPl and CmedSNMP2). Most of the genes have small dN/dS ratio (<0.2), thus, indicate that purifying selection are act on these SNMP genes. By using Bac-to-Bac system we expressed ChsupSNMP1and ChsupSNMP2in Tn cells, cellular immunofluorescence showed that the two SNMPs both have intracellular N-termini and C-termini. The distribution patterns of SNMP genes in different tissues of adult moths were examined using RT-PCR and quantitative real-time PCR. Although the four SNMPs are expressed not only in antennae but also in non-olfactory tissues such as wings, legs and body, the relative transcription level shows these genes are highly enriched in antennae. On the other hand, relative expression levels of ChsupSNMPl and ChsupSNMPl at different developmental stages of Ch. suppressalis were subsequently examined. Transcript levels of both two ChsupSNMPs were very low in the embryos, larvae and early-stage pupae, but increased significantly since the late-stage pupae and reached their maximum at adult stage. We also found that in pupae, as well as in adults, ChsupSNMPl transcripts were significantly higher than ChsupSNMP2.4) We isolated a putative NADPH-cytochrome P450reductase gene from Ch. suppressalis, named it CsCPR. qPCR data showed that noticeably abundant CsCPR mRNA transcripts were detected in larval midgut and in antennae of adults, and the expression levels were significantly higher than other tissues in larvae and adults, respectively. Since CPR has a function to provided electrons to various P450s, it could be involved in the odorants and volatile xenobiotics clearance in antennae, thus protect the insect olfactory receptor neurones. We heterologously expressed CsCPR in E. coli, and generated a soluble protein with the cytpchrome c-reductase activity. The Km value of the enzyme was35.71±5.37μM/min/mg.In conclusion, olfactory genes of C. medinalis and Ch. suppressalis, including OBPs,Orco. SNMPs and CPR. were studied in this dissertation. These genes are basic components of the olfactory signal transduction cascade, and are central parts of the olfactory network. Results of our investigation could provide valuable information for understanding the basic mechanism of insect olfaction, and for finding appropriate targets to develop novel environmental friendly attractants and repellents.
Keywords/Search Tags:Cnaphalocrocis medinalis, Chilo suppressalis, olfactory, gene, odorant-binding protein, olfactory co-receptor, sensory neuron membrane protein, NADPH-cytochrome P450reductase
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