| The tea green leafhopper [Empoasca(Matsumurasca)onukii Matsuda] is a major pest of tea trees,and chemical control is the most important preventive measure of E.onukii.However,the excessive use of pesticides causes serious pesticide residue problems,which endangers human health and causes environmental pollution.Insects use highly sensitive olfactory system to locate hosts,foraging,spawning and reproduce.A variety of olfactoryrelated receptors are involved in the olfactory recognition process of insects,including odorant receptors(ORs),ionotropic receptors(IRs),gustatory receptors(GRs).At present,the research on the olfactory-related genes of most insects were limited to the identification,and the functional exploration of them has a key role in further molecular means to pests control.In this study,Eonu IR25 a and Eonu GR1 were cloned,and the gene expression of them in different tissues of male and female E.onukii was analyzed by q RT-PCR.Differences in behavioral tropism to tea trees volatiles before and after Eonu IR25 a and Eonu GR1 interfered were verified by RNA interference and behavioral assays in E.onukii.The study of the function of olfactory-related receptor genes in the recognition of tea trees volatiles provides a basis for the targeted development of behavior regulator for the ecological control in E.onukii.The main findings are as follows:1.Eonu IR25 a and Eonu GR1 were cloned,the CDS length of Eonu IR25 a was 1266 bp and it encoded a 48.87 k D protein.There are 4 transmembrane domains in Eonu IR25 protein,containing the conserved structural domains of Lig＿chan(positions 41-350)and SBP＿bac＿3(positions 1-317),and it is a hydrophilic protein with theoretical pl of 6.50.The CDS length of Eonu GR1 was 1066 bp and it encoded a 40.51 k D protein.There are 4 transmembrane domains in Eonu GR1 protein,containing the conserved structural domains of 7tm＿7(positions 1-346),and it is a hydrophobic protein with theoretical pl of 9.33.2.The q RT-PCR results showed that the gene expression of Eonu IR25 a was significantly higher in the antennae of male and female than other tissues,and it was the least in the female legs.Eonu IR25 a is expressed in the female antennae was 4-7 times higher than female legs and thorax.There was no differences between male and female antennae of Eonu IR25 a,but there was highly significantly differences between different tissues in E.onukii.Eonu GR1 is expressed in the female abdomen was significantly higher than other tissues,and the gene expression was 21.3 and 2.7 times higher than female legs and antennae.The gene expression of Eonu GR1 in the abdomen was 7 times higher than head and thorax,and there was highly significant differences between different tissues of Eonu GR1.3.The results of multiple sequence alignment and phylogenetic tree showed that Eonu IR25 a clustered in the smallest branch with Homalodisca vitripennis Hvit IR25 a and Hvit IR25a-like.The sequence similarity between Eonu IR25 a and other hemiptera IRs ranged from 30-40%,and the similarity between Eonu IR25 a and Hvit IR25 a was 36.52%.The similarity between Eonu GR1 and other hemiptera GRs ranged from 15-20%,and the similarity between Eonu GR1 and Hvit GR1 was 17.14%.Eonu GR1 clustered with GR22 and GR63 a of hemipterans.4.The results in the field and indoors experiments showed that E.onukii adults were more attracted to the volatiles 1-phenylethanol.The results of RNAi showed there were the highest interference efficiency at 3 h in Eonu IR25 a and Eonu GR1,and the relative expression of the target genes was the lowest.The interference efficiency of root soak was significantly higher than foliar spray,and the effect was stable.After interfering with Eonu IR25 a,the response to 1-phenylethanol at concentrations of 0.1,10 and 100 μL/m L decreased with26.67%,30% and 30%,respectively,which speculated Eonu IR25 a may play a role in the recognition of 1-phenylethanol in E.onukii.After interfering with Eonu GR1,the response to phenylethylaldehyde at concentrations of 10 and 100 μL/m L were significantly decreased with 30%,which speculated Eonu GR1 may be involved in the recognition of phenylacetaldehyde in E.onukii. |
