Study On Calcium Channel-related Genes Of Carmine Spider Mite,Tetranychus Cinnabarinus Under Scopoletin Stress

Through screening a series of compounds,our laboratory found that scopoletin,a coumarin compound,is an important secondary plant metabolite and phytogenous acaricidal compound with excellent contact-killing,systemic,repellent and oviposition inhibition activities against Tetranychus cinnabarinus.However,the acaricidal mechanism of scopoletin remains unclear.Therefore,characterization of the molecular mechanism or the molecular targets of scopoletin against Tetranychus cinnabarinus?Boisduval?will benefit a lot to use scopoletin as a template for molecular optimization to develop new green acaricides.To reveal the molecular mechanism and search for candidate molecular targets of scopoletin against T.cinnabarinus,we hypothesized that Ca²⁺channels are important in the acaricidal mechanism of scopoletin.We have obtained the following research results:1.We obtained and verified the RNA-Seq results,and validated the main biological process of response genes involved in the response genes of scopoletin against T.cinnabarinus.A total of52,496,305 clean sequence reads were generated from scopoletin-treated mites and 52,286,859 clean sequence reads were generated from solvent-treated mites.Differentially expressed genes at 24 h and48 h were 15481 and 15411,respectively.According to RPKM values,70 and 102 genes were identified as significantly differentially expressed genes between scopoletin-and solvent-treated mites for 24 and 48 h,respectively.Moreover,qPCR confirmed the accuracy of the results of RNA-Seq,indicating that the 15 genes of T.cinnabarinus were indeed involved in the response to scopoletin.Gene Ontology?GO?function enrichment analysis revealed that the major biological processes involved in significantly differentially genes,which includes cellular physiology,metabolic processes,single biological processes,cell apoptosis,ligation and catalytic activity,ion transmembrane transport,neurve conduction and so on.KEGG pathway enrichment analysis lists the top 20 biochemical metabolic pathways and signal transduction pathways at 24 h and 48 h post-treatment,of which phosphatidylinositol signaling system,calcium signaling pathway and MAPK signaling pathway are the most representative biochemical pathways.The genes contained in these GO and KEGG entries are likely to be closely linked to the the mechanism of scopoletin against mites.2.We obtained significantly differentially genes related to mite metabolism and calcium signaling,and determined the expression changes of three calcium channel related genes after scopoletin treatment.Through RPKM and Noiseq algorithm,we screened out the total number of172 significantly differentially expressed genes from scopoletin treated mites and 18 of them identified as common differentially expressed genes for both time points.In this study,we obtained three genes closely related to calcium channels,such as guanylate kinase?GUK?,Bcl-2 protein?BAG?and G protein-coupled neuropeptide receptor?GPCR?,which are important signal transduction proteins.Specific expression detection shows that scopoletin treatment upregulates the expression levels of TcGPCR and downregulates the expression levels of TcBAG and TcGUK,which could be used as key genes for the study on the mechanism of scopoletin against mites.3.We cloned and analyzed the cDNA sequence and expression pattern of TcGPCR,TcBAG and TcGUK.We obtained the cDNA sequences of TcGPCR,TcBAG and TcGUK by cloning and were deposited in GenBank under the following accession numbers:KY660540,KY660539 and KY660538,respectively.In addition,we found that they have high similarity with homologous genes of other species through sequence alignment,phylogenetic tree analysis,protein transmembrane structure and domain prediction,which are closely related to calcium channel and are calcium channel related genes.Specifically,the calcium channel-related genes?TcGPCR,TcBAG,and TcGUK?were significantly highly expressed during the larval and nymphal stages compared with other developmental stages.4.Identification of molecular targets of scopoletin against T.cinnabarinus.All 3 genes were successfully silenced using RNAi.The result of RNAi indicated that downregulation of TcGPCR decreased acaricidal activity of scopoletin,and downregulation of TcBAG and TcGUK enhanced acaricidal activity of scopoletin,suggesting that the three calcium channel-related genes are crucial in the acaricidal mechanism of scopoletin against T.cinnabarinus.What is more,functional expression in Chinese hamster ovary cells and calcium reporter assays strongly showed that scopoletin and coumarin derivatives had agonistic activity on TcGPCR.Meanwhile,our results demonstrated that substituent group,especially on the C-6,C-7 sites,can enhance the agonistic activity of coumarins on TcGPCR,which suggested that coumarin compounds may have similar molecular target in T.cinnabarinus.The current findings make it clear that the GPCR-sensitive calcium release channel may be the primary target of scopoletin in T.cinnabarinus;at the same time,other calcium channel related genes,such as TcBAG and TcGUK,can not be excluded as the target of scopoletin in mites.Taken all together,these results suggested that the action mode of scopoletin may be through the regulation of the expression levels of calcium channel-related genes,thereby regulating calcium channel activity and eventually inducing cell calcium overload to kill mites.