| Corolado potato beetle(CPB), Leptinotarsa decemlineata(Say), is the most destructive pest of potato, and is a major quarantine pest in China. As chemical management is the main control methods for CPB, CPB has evoled resistance to most of registered pesticides. An alternative techniques of chemical control is under urgent need at present. RNA is a potential pest control method, which is high specifity, environmental friendly. However, RN A has not been applied in fields because of low stability of dsRNA, unclarified RNA s ignal transduction pathway and few researh for RNAi mechanism in insects.In this study, 6 RNA-core componemt genes have been cloned by RT-PCR, analyzed by multiple alignment and phylogenetic tree. Temporal and spatial expression patern were characterized by qPCR. Genetic function in RNAiwere identified by RN A. The main results were as follows:1. By using RNA-core component genes in Drosophila melanogaster and Tribolium castaneum and Blast, 2 Sid-1,2 Dicer2and2 Ago2 cDN A were found in CPB’s transcriptome and genome. By a series of molecular techniques, including RNA extraction, reverse transcription and PCR, 2 complete ORF of Sid-1s, 2 complete ORF of Dicer2 s and 2 partial coding sequences of Ago2 s were cloned. By multiple alignment of Sid-1 sequences of CPB, fruitfly and red flour beetle, 2 Sid-1s in CPB contain typical Sid-1 structure, including signal peptide, 4 conserved motifs and 11 transmembranes, indicating 2 Sid-1s in CPB may function as other Sid-1s in insects. By phylogenetic clustered analysis, 2 Sid-1 clustered with red flour beetle’s TcSid-1a and TcSid-1c and has highest similarity, respectively. Therefore, 2 LdSid-1s were nominated as LdSid-1a and LdSid-1c, respectively. 2 Dicer-2 and 2 Ago-2 were analyzed by multiple alignment and nominated before.2. Temporal and spatial expression patern were analyzed by qPCR, results were as below,(1) Besides Ld Ago2 b, 6 RNA-core component genes have similar expression pattern: relative expression level increased as larvae grows and get highest as larvae grow to 4t h instar. Moreover, LdAgo2 b was expressed highly in 0 hours and 72 hours old 4t h instar, but has trace level in other stage. These results indicate these genes were involved in important physiological function in elder stage larvae.(2) Spatial expression patternwere detected in various tissues in 4t h instar larvae and reproductive organs in adults. Results indicate that besides Ld Ago2 b, 6 genes were expressed widely in all tissues and have d ifferent expression pattern, even though homologues.3. By ds RNA-feeding methods, RNA-core component genes in CPB were characterized. Firstly, suppresing expression of RNA-core component genes in CPB by feeding dsRNA did not affect larvae’s phenotypes. Therefore, ds ATPase E was applied in vivo to assess the involvement ofgenes in the RNA pathway. dsATPaseE’s 4t h instar LC50 was the concentration of 1/883 of ds ATPase E-expressed bacteria. By pretreatment of 9 groups:(1)ds LdDicer2 a and LdDicer2 b,(2) LdAgo2 aand LdAgo2b,(3) LdSid-1aand LdSid-1c,(4) LdDicer2 a,(5) LdDicer2 b,(6) LdAgo2 a,(7) LdAgo2 b,(8) LdSid-1aand(9) LdSid-1c,1/10, 1/100 and 1/1000 concentration of dsATPaseE was applied three days later. We found that treatment of dsLdAgo2 and dsLd Dicer2 could affect lethal effect of ds ATPase E by decreasing RNA effect of dsATPase E. In contrast, treatment of(1) dsSid-1a,(2) dsSid-1c, or(3) dsSid-1 and dsSid-1ccould not affect lethal effect of any concentration of ds ATPase E compared to treatment of dsegfp, although expression level of LdSid-1a and LdSid-1c was detected by qPCR and suppressed. All these results indicate LdSid-1aand LdSid-1ccould not affect RNA like LdDicer2 a, LdDicer2 b, LdAgo2 aand LdAgo2b. |
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