| The Asian gypsy moth, Lymantria dispar Linnaeus, is one of the key defoliators worldwide. It is an omnivorous insect of large food intake, which causes the great economic losses. At present chemical pesticides are mainly used to control the Asian gypsy moth. The resistance of gypsy moth to chemical pesticides easily occurs for the frequent usage of chemical pesticides. It is urgent to identify the new molecular targets to develop the novel pesticides. The genes in insects involved the growth, reproduction, nutrition, movement and so on, are usually the ideal targets of the pesticides. GPCR in insects are widely concerned because it involves in the important physical functions and response to the Endogenous and Exogenous compounds. It is great significant to explore the functions of genes in GPCR superfamily for identifying the new targets for screening novel pesticides.In this paper, the Asian gypsy moth is selected as our object to deeply study the GPCRs in insects, which are the possible target of the pesticides. Firstly, the transcriptomes were constructed by the high through put sequencing technology, the Asian gypsy moth was treated with sublethal dose of cyantraniliprole which is one of the new type pesticide targeting ryanodine receptor in insects. GPCRs family genes in the Asian gypsy moth were screened by analyzing the differentially expressed genes. The functions of OA1, Mthl, Mth2 genes and the mechanism of response of these genes to pesticide stress were specially analyzed. The main results are as the following:1. The transcriptomes data base of the 2th instar Asian gypsy moth treated by cyantraniliprole (LC20=0.187 mg/L) and the control (acetone) were constructed; 26.31 Gb data were obtained and Q30 base was over 87.30%. Total 40830 of Unigene were obtained and the length of 9529 among them was over 1kb; 18199 genes were annotated in Nr protein bank, 10532 genes were annotated in SwissProt protein bank,4826 genes were annotated in KEGG protein bank,4861 genes were annotated in COG protein bank,10133 genes were annotated in GO protein bank; Total 18260 functional genes were obtained2.27 GPCRs genes in the Asian gypsy moth were identified from the constructed transcriptomes data base. Analyzed with the RT-qPCR method 18 GPCRs genes were response to the stress of deltamethrin, omethoate, Carbaryl and cyantraniliprole. The results showed that Unigene21812,Unigene32849,Unigene40527,Unigene42365,Unigene47827,Unigene48286 and Unigene 15819 were mainly upregulated, the others are downregulated under the stress of dehamethrin;Unigene39352,CL3275.Contig1,Unigene40527,Unigene42365,Unigene47827,Un igene48286,Unigene15819 were mainly upregulated under the stress of Carbaryl; 8 GPCRs genes(Unigene44172,Unigene46527,Unigene46885,CL8307.Contigl,Unigene5315,Unigene97 75,Unigene 11368,Unigene15505)were downregulated and 10 GPCRs genes (Unigene21812,Unigene32849,Unigene39352,CL3275.Contigl,Unigene40527,Unigene42365, Unigene47827,Unigene48286,Unigene4933,Unigene15819)were upregulated under the stress of omethoate;14 GPCRs genes were upregulated under the stress of cyantraniliprole; the above results indicated that GPCRs possibly involved in the response to the stress of pesticides. In this paper the full length sequence of 3 GPCRs genes (Unigene44172,Unigene46527,Unigene46885)were obtained by cloning, which were ocular albinism type gene (LdOAl), longevity genes (LdMthl, LdMth2)of GPCR family with the prediction of protein conserved domain. The length of gene ORF was 453～1563 bp, and coded with 150-520 amino acid; the molecular weight was 17.54-59.18 kDa, pI was 9.76-10.07, and all was the basic protein.3. Analyzed with the RT-qPCR method LdOAl, LdMthl and LdMth2 specially expressed in the growth stage. The results showed that 3 GPCRs all expressed in each growth stage of the Asian gypsy moth and expressed at high level in the stage of egg. The expression level of LdOAl and LdMthl were at the peak in the stage of adult male and female respectively, the result indicated LdOAl, LdMthl and LdMth2 expressed specially in the stage of growth.4. The gene silenced Asian gypsy moth was prepared by micro-injection of the special double-stranded RNAs of LdOAl, LdMthl and LdMth2 with the RNAi Technique. The transcriptional level of mRNA at different time and nutrient utilization index were measured, the results showed that the best silencing effect of LdOAl and LdMth2 was at 120h, which were 95.18% and 82.00% respectively and higher than that of control (GFP); the silencing effect of LdMthl was 97.96% at 96h, and obvious at every other time except 24h and 48h. Compared with the control group (ddH2O and GFP) the treated group micro-injected LdOAl and LdMthl showed no significant difference on food intake, figure and food utilization after being fed 8 days; the relative food intake ratio was 7.19% and 1.83% lower than that of control group (dsRNAGFP) respectively, which affected the growth of Asian gypsy moth larvae, but no obvious mortality occurred. The bioassay was conducted to analyze the sensitivity of gene silenced Asian gypsy moth to pesticides, the results showed that sensitivity of Asian gypsy moth to pesticides was greatly improved when the targeted gene was silenced. From the treatment of deltamethrin we found that, compared with control group micro-injected dsRNA GFP the mortality of treated group with dsRNALdOAl, dsRNALdMthl and dsRNALdMth2 was increased by 20.42%,39.20%,48.30% respectively within 120h; the inhibitory rate of transcriptional level of that treated with LdOAl, LdMthl and LdMth2 was 98.53%,99.85%, 99.85% respectively; however, the sensitivity of the Asian gypsy moth larvae treated with dsRNALdOAl, dsRNALdMthl and dsRNALdMth2 to cyantraniliprole was increased by 15.96%, 56.67%,8.49% respectively.5. The six transformed D. melanogaster were obtained by being micro-injected LdOAl, LdMthl and LdMth2 into 2# and 3# chromosome of D. melanogaster, which was named attp2#-LdOAl, attp3#-LdOAl, attp2#-LdMthl and attp3#- LdMthl, attp2#-LdMthland attp3#-LdMth2 respectively. Compared with the control the average life of the transformed D. melanogaster was increased by 51.03%-69.89%.The sensitivity of the transformed D. melanogaster to pesticides was analyzed, the results indicated that attp2#-LdOAl, attp3#-LdOAl, attp2#-LdMthl and attp2#-LdMth2 were sensitive to deltamethrin; however, the resistance of attp3#-LdMthl, attp3#-LdMth2 to deltamethrin was increased to 1.65 and 1.50 fold. In order to study the response of downstream genes in the transformed D. melanogaster to pesticides, which expressed GPCR of the Asian gypsy moth, the expression level of HSP, GST, CYP in the transformed D. melanogaster stressed by low concentration of deltamethrin was analyzed with RT-qPCR technique, the results showed that GST regulated by Mth gene was in response to the stress of deltamethrin; the transcriptional level of GST family was 1.09-17912.35 fold,1.10-2155.29,1.30-647.17 and 1.25-1646.67 fold higher than that of non-transformed D. melanogaster, (attp2#-LdMthl), (attp3#-LdMthl), (attp2#-LdMth2) and attp3#-LdMth2 respectively; however, CYP6a8, CYP6a9, CYP4e2, hsp23, hsp26, hsc.70-5 (attp2#-LdMthl and attp2#-LdMth2),were upregulated, and CYP6wl, hsp22, hsp26, hsc70-5(attp3#-LdMthl and attp3#-LdMth2) were mainly upregulated; the genes of CYP, HSP, GST in 3# transformed D. melanogaster expressing LdOA1 mainly expressed by inducement, their expression level was greatly higher than that of non-transformed D. melanogaster; the genes of CYP and HSP in 2# transformed D. melanogaster expressing LdOA1 were mainly downregulated, the expression level of GST family was greatly higher than that of non-transformed D. melanogaster. |
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