Cloning Of Ryanodine Receptor From Plutella Xylostella And Spodoptera Exigua And Molecular Pharmacology

Some researchers have been interested in the synthesis of chemicals targeting at insect ryanodinereceptor (RyR) or biochemical and molecular function characterization of insect RyR, sinceflubendiamide and chlorantraniliprole were founded and developed. The two diamides have highactivities against Lepidoptera insects. However, the action sites of which were unclear at present. Thediamond back moth Plutella xylostella and beet armyworm Spodoptera exigua fat bodies cell lineIOZCAS-Spex-II were recognized as objects in this paper. The RyR cDNA of the two insects werecloned, and the effects of diamides on their RyR relative expression abundance were carried out.Conclusions are as follows:1) The Px-RyR cDNA total sequence of15748bp was assembled by overlapping all theamplified fragments sequences. The final full-length Px-RyR cDNA contained a5'-UTR of267bp, a3'-UTR of109bp and a full open reading frame (ORF) of15372bp.5123amino acidsresidues were predicted by the ORF, and the molecular weight was579.5kDa, the pI was5.45.There were six essential transmembrane (TM) segments predicted from the secondary structure ofthe deduced amino acid sequence. And a motif, GVRAGGGIGD, was identified, which had beenshown to constitute a part of pore-forming segments of the Ca2+release channels. In addition, thefour repeated sequences were found in Px-RyR, had averaging33%identity in Px-RyR. TheBLAST results showed that the deduced amino acids sequence of Px-RyR were very similar to thefunctionally verified RyRs from tobacco budworm Heliothis virescens (92%), silkworm Bombyxmori (92%), fruitfly Drosophila melanogaster (78%), Mus musculus RyR1(45%), Mus musculusRyR2(47%), Mus musculus RyR3(46%).Relative expression of ryanidine receptor mRNA from Plutella xylostella was carried out byquantitive RT-PCR method. The result showed that ryanodine, fluebndiamide, chlorantraniliprole andcyantraniliprole can promote the expression of Px-RyR. In the uncontinuous treatments, the larvae wereraised24h on leaves with insecticides, then raised further24h on cabbage leaves without insecticides.The Px-RyR relative expression abundance changed depending on the different concentration of4insecticides. The ryanodine receptor relative expression from resistance strain was the5.79-fold tosusceptible strain. However, emamectin benzoate and chlorfenapyr have no effects on the Px-RyRrelative expression.2) The ORF sequence of Se-RyR cDNA contained15372bp,5118amino acids residues werepredicted by the ORF, and the molecular weight was578.7kDa, the pI was5.44. And othercharacterizations are the same as Px-RyR.After treatment24h, the Se-RyR relative expression abundance increased with the increasingchlorantraniliprole concentrations (0.1nM,1nM,10nM,1M,100M). And the same results wereshowed in the uncontinuous treatments. Statistical analysis showed that there was a linear positivecorrelation between the logarithm of chlorantraniliprole concentrations and the Se-RyR relative expression.Given the structural and functional importance of RyR from Plutella xylostella and Spodopteraexigua in this work, and effects of diamides on the RyR relative expression were discussed at the firsttime. The results could provide the basis for further interaction study of resisitance, and were helpful toindicate the mechanism of diamides insecticides and screen compounds targeting at RyR, and to providea basis for further study of diamides resisitance in the future.