Molecular Analysis And Functional Characterization Of TRP Channels In Brown Planthopper,Nilaparvata Lugens(Hemiptera:Delphacidae)

The Transient Receptor Potential(TRP)superfamily of ion channels comprises a collection of cation channels conserved from worms to flies and humans,and TRPs are activated through a wide variety of mechanisms and participate in virtually every sensory modality such as touch,smell,hearing,proprioception,etc.Latest research shows that the protein complex of Nanchung and Inactive which belongs to TRPV subfamily of insect was the target of pymetrozine(Nesterov et al.,2015),this was the first report of the TRP channels involved in pesticide molecular target.Drosophila melanogaster as a model organism,the research of TRP channels was relatively more,but there was few researches in agricultural pests.In this paper,we chose the brown planthopper,Nilaparvala lugens(Hemiptera:Delphacidae),one of the main pests that cause serious damage to rice production in Asia,as object,have cloned all(13)TRP channel genes in N.lugens,using quantitative PCR technique has proven the age and tissue expression pattern of TRP channel genes in N.lugens,and using RNAi technique to studied the influence of survival,motor coordination and touch response when the RNA silenced in N.lugens.1.Cloning and sequence analyses of TRP channel genes from brown planthopperBased on the amino acid sequences of TRP channel proteins from Drosophila melanogaster,which was obtained from GenBank,TBLASTN searches of the N.lugens genomic and transcriptomic databases,we have identified 13 genes encoding TRP channel deducing proteins.And then we used the technique of PCR and RACE confirmed the full-length cDNA sequences of the 13 TRP channel genes based one the deducing protein sequences which was obtained from transcriptomic databases.These 13 TRP channel genes can be divided into six subfamilies,TRPC,TRPA,TRPV,TRPN,TRPM and TRPML subfamily.Meanwhile,the evolution of TRPA subfamily was different between fruit fly and brown planthopper,pyrexia homologous gene of Drosophila was not exit in brown planthopper,but have two TRPA5 genes,NlTRPA5-1 and NlTRPA5-2.At the same time,we found that the genes of TRPC,TRPV,TRPN,TRPM,TRPML subfamilies more conservative during evolution in different species,which also has a high homology between the three genes of brown planthopper TRPC subfamily.The TRPA subfamily genes are not conserved during evolution besides NlTRPA1 and NlWtrw.Besides the NlPain and NlWtrw,the others have intron/exon structure,NITRPM has the largest number of exons in the region of CDS.TRP channels of brown planthopper have six transmembrane domains and one pore structure located in the fifth and sixth transmembrane domains,the C-terminal of the genes in TRPC,TRPN,TRPM subfamilies have TRP domains(EWKFAR and LPPPFN),the ankyrin repeat in the N-terminal was different in different genes,NlTRPL,NlWtrw,NkTRPA5-1,NlIav and NITRPM also have coiled coil region in C-terminal.2.Expression pattern of TRP channel genes in brown planthopperWe had studied the expression patterns of the 13 TRP channel genes which were cloned in the brown planthopper by qPCR method.From the experimental results,NITRP and NITRPL was highly expressed in head,which was coincide with the function of visual.The expression of NITRPy was highly expressed in antenna and head,and the expression level of male was higher than female.In TRPA subfamily,NITRPA1 highly expressed in the antennae,other genes in this subfamily also can be detected in all tissues,but the expression pattern was different;in the developmental expression pattern,they all highly expressed in adult stage,while the egg stage was relatively lower expressed.NINan and Nllav which belongs to TRPV subfamily,they all highly expressed in antennae,and the expression in male was higher than female in the adult stage,NlNan was highly expressed in adult stage,but the expression of NlIav was mainly in 1 instar nymphs.NlNompC which belongs to TRPN subfamily was mainly expressed in the abdomen,and it was highly expressed in adult stage.NlTRPM can be detected in all tissues and was higher expressed in abdomen and thorax.NITRPML was highly expressed in the short-wing brown planthopper,mainly expressed in the abdomen of N.lugens.3.Nilaparvata lugens TRP channel gene silencing effect on survivalWe injected the dsRNA of TRP channel genes into brown planthopper third instar nymphs respectively,and then we used the qPCR to detcted the effect of silencing.Unfortunately,NINan,NlTRPA1,NlWtrw and NITRPA5-2 was not silenced effectively,the others was silenced significantly.the brown planthopper which NlNompC silenced have low survival rate,they showed significant movement disorders,the legs couldn't stretch or bent normaly,and the angle of femur and tibial in hind legs was significantly greater than control.Meanwhile,NlNompC silenced would lead to a decline in gentle touch response,the control of ds-GFP injected could escaped when we gentle touched them.The other eight genes silenced couldn't lead the brown planthopper to dead.