Molecular Mechanism Of Flubendiamide And NK130102on Ostrinia Furnacalis

NK130102is a novel phthalic amide insecticide. In order to investigate the mechanism ofthis compound, transcriptome sequencing was used to evaluate changes in the globle geneexpression of O. furnacalis treated with NK130102and flubendiamide. Based on thetranscriptome sequencing results, the OfRyR cDNA was cloned. The effects of differentinsecticides and different concentration of flubendiamide on OfRyR cDNA relative expressionabundance wre carried out. We also cloned the gDNA of OfRyR based on the OfRyR cDNA.Conclusions are as follows:1) The LC50values of NK130102and flubendiamide against Ostrinia furnacalis were0.2and0.17mg g-1respectively, and the LC95values of two insecticeides against Ostrinia furnacalis were8.8and3.8μg g-1respectively. The ryanodine receptor (RyR) gene and sarco/endoplasmicreticulum calcium ATPase gene in the calcium signaling pathway were up-regulated for3.7and2.8folds, and the myosin, actin, muscle M-line assembly protein unc-89, titin genes involved invascular smooth muscle contraction were up-regulated for7.4,2.8,26.9and3.7folds in O.furnacalis treated with NK130102at the concentration of8.8mg g-1for48h. Therefore, we coulddeduce that NK130102acting on the calcium release channel, reduced the continuous Ca2+releasefrom calcium pump, and the increased Ca2+in cytoplasm caused the persistent muscle contraction.In particular, the insecticide targets and metabolism genes will facilitate the designing of newcompounds based on the insecticide target.2) Based on the transcriptome sequencing, we cloned and characterized the full-length RyRcDNA from Ostrinia furnacalis. This cDNA contained an ORF of15324bp encoding a protein of5108amino acid residues, which show79–97%amino acid identity with other insect RyRisoforms and share the greatest identity with Cnaphalocrocis medinalis RyR (97%). Quantitativereal-time PCR showed that the OfRyR was expressed at the lowest level in egg and the highestlevel in adult. The relative expression levels of OfRyR in first, third and fifth-instar larva were1.19~1.99times of that in egg. Moreover, two alternative splicing sites were identified in theOfRyR gene. One pair of mutually exclusive exons (a/b) were present in the central part of thepredicted second SPRY domain, and an optional exon (c) was located between the third and fourthRyR domains. Diagnostic PCR showed that exons a and b existed in all developmental stages ofOfRyR cDNA, but exon c was not detected in the egg cDNA. And the usage frequencies of theseexons were different among different developmental stages.3) Morphological study demonstrated that the weight of O. furnacalis treated withflubendiamide and NK130102were much lighter than those treated with emamectin benzoate andDMSO. Flubendiamide and NK130102also caused the toxic symptom of antifeedant, vomiting,contract and shortening. Real-time fluorescent quantitative PCR showed the relative expressionquantity of RyR cDNA was increased when O. furnacalis treated with flubendiamide andNK130102, while emamectin benzoate has no effect on the OfRyR expression. The relative expression quantity of RyR cDNA from O. furnacalis treated with different concentration offlubendiamide was also carried out. The OfRyR cDNA increased with the increasingflubendiamide concentrations after treatment24h, and flubendiamide at lower concentrations canlead to continuing rise over time. The biggeat raise of OfRyR cDNA was2.96-fold than controlwhen Flubendiamide at1μg/g for48h.4) We cloned the gDNA of OfRyR based on OfRyR cDNA. The full length of93575bpOfRyR gDNA contains95exons, the25and26exons are a pair of exclusive exons and the59exon is an optional exon. The exon/intron boundaries assigned follow the GT/AG rule.In conclusion, transcriptome sequencing data provide useful information in understanding themechanisms of novel insecticide NK130102. We cloned cDNAand gDNA of OfRyR and analysedthe effects of diamides on the expression of OfRyR, which further clarify the mechanism ofdiamide insecticides. The results will facilitate the designing of new compounds based on theinsecticide targets and could provide the basis for further study of resisitance to diamideinsecticides.