The Study Of Para Sodium Channel,NlSC1Ion Channel And Three Key Genes On The Biosynthetic And Metabolic Pathways Of Juvenile Hormone In Brown Planthopper

Rice planthopper, a primary pest in Asia, brought severe reduction of rice production, and endangers the food security by sucks fluid from rice or spreading rice viruses. In recent decades, insecticides have been playing an important role in rice planthopper control, but there are some successive reports about the insecticides resistance from rice planthopper. In order to understand the mechanisms of insecticides resistance, and to screen new chemicals for old targets or to explore new targets, voltage-gated sodium channel, DSC1ortholog-a new cation ion channel, and three key genes on the biosynthetic and metabolic pathways of juvenile hormone(JH) in brown planthopper were studied.DSC1is a voltage-gated cation channel with close structural and evolutionary relationship to voltage-gated sodium channel, but has different function. It was used as query sequence for searching its orthologs from all genomes published in GenBank, the result showed that its orthologs distributed in a few phylum, including Arthropoda, Mollusca, Cnidaria, Hemichordata and Echinodermata, and most of them are from Arthropoda, but none in vertebrates. Two orthologs from the brown planthopper (Nilaparvata lugens) and the silkworm (Bombyx mori) were cloned and sequenced, named NISC1and BmSCl, respectively, their cDNA have lots of conserved exons and several unique optional exons. NlSC1and BmSC1have about50%identity with DSC1, and their transcripts were most abundant in the heads and antennae in adults. Hydropathy profile analysis indicated that all DSC1orthologs contain an unique and conserved DEEA motif which is critical for ion selectivity in the pore regions except a few poorly annotated sequences, instead of the EEEE or EEDD motif in classical calcium channels or the DEKA motif in sodium channels. They also have an unique and conserved MFL motif in the loop linking domains III and IV. Phylogenetic analyses revealed that DSC1and its orthologs form a separate group from the classical voltage-gated sodium and calcium channels. These results fully confirm that DSC1and its orthologs do constitute a distinct family of cation channels. The absence in vertebrates implies that the DSC1/BSC1-family channels might be good targets for the development of new insect-specific cation channel blockers for pest control. Insect sodium channels are targets for DTT, pyrethrins and pyrethroids. Para protein from Drosophila melanogaster was used for searching its ortholog in the brown planthopper genome, a ortholog was found, and was cloned through RT-PCR and RACE. The longest para full-length cDNA is6215bp encoding2084amino acid. Sequence alignments indicated that6RNA alternative splicing sites and10RNA editing sites were found in its ORF,35potential phosphorylation sites for cyclic AMP-dependent protein kinase (PKA) and47phosphorylation sites for protein kinase C(PKC) were predicted, and eight potential N-linked glycosylation sites predict from its proteins sequence. The results from qPCR showed that para from brown planthopper is abundant in its antenna and head. Now, five types of para ORF are testing for studying its electrophysiology, the result will be helpful for understand the function of the RNA alternative splicing sites and RNA editing sites.JHs play important roles in the growth, development, metamorphosis and reproduction of insects, in order to understand the biosynthetic and metabolic pathways of juvenile hormone in brown planthopper, and screen inhibitors for these enzymes as insecticides, juvenile hormone epoxide hydrolase (JHEH), juvenile hormone acid methyl transferase (JHAMT) and farnesyl pyrophosphate synthase (FPPS)were cloned by RT-PCR and RACE based the transcriptome, their full-length cDNA are2114bp,1144bp and2114bp encoding454,279and393amino acid, respectively. The three genes were expressed in insect cells, the results from SDS-PAGE and Western Blotting revealed that they expressed the right size protein with approximate52,32and45kDa. The analysis from qPCR indicated:the relative expression of JHEH reaches its peak one day before molting or eclosion, and reaches its lowest level; that of JHAMT has no significant change, but reach its highest one day before eclosion, after eclosion, it is increasing gradually; that of FPPS is reach moderate level, and reach its highest one day before eclosion, and maintain moderate level. Homologies modeling of the three proteins were performed by using SWISS-MODEL, the results provide structural base for screen their specific inhibitors.