Analysis Of MRNA-Seq And The Response Genes Of Carmine Spider Mite,Tetranychus Cinnabarinus Under Curcumin Stress

By screening a series of compounds,our laboratory found that curcumin,a plant derived compound that originated from the herbal plant Curcuma longa,showed promising acaricidal activity and great potential to be developed into a green acaricide.Therefore,characterization of the molecular mechanism or identification of the molecular targets of curcumin against Tetranychus cinnabarinus(Boisduval)will benefit a lot to use curcumin as a template for molecular optimization to develop new green acaricides.At present,although some progresses have been made on the acaricidal mechanism of curcumin,the molecular mechanism or molecular targets of curcumin against T.cinnabarinus remains largely unknown.Also,the lack of genetic information on this organism and response genes closely related to acaricidal mechanism of curcumin is a major obstacle in this field.In this study,an improved digital gene expression profile sequencing technology was used to explore the molecular mechanism and stress response genes,expending the gene information resources of this species,pointing out the direction for the acaricidal mechanism of curcumin and enriching the research on insecticide mechanism of natural active substances.The main research results are as follows:1.The LC50 of curcumin against female adults of T.cinnabarinus was 2.642 mg/m L,and the curcumin-exposed mites showed similar neurotoxic symptoms.The bioassay result after 48 h treatment indicated that the toxicity regression equation was y =-0.278 + 0.659 x with median lethal concentration 2.642 mg/m L and 95% confidence interval 1.680~6.082 mg/m L.After treating with curcumin by slide dip and leaf dish spray,we observed that the poisoning symptoms of T.cinnabarinus can be divided into the following four phases: stationary phase(0 h-4 h);recovery and exciting period(4 h-48 h);paralysis coma period(48 h-72 h);death period(72 h-144 h).It has some similarities with the poisoning symptoms of nerve agents.According to the poisoning symptoms,24 h and 48 h were considered as the most important time points for the next sequencing experiment.2.We obtained and verified the digital gene expression profile sequencing results,and validated the main biological process of response genes involved in after treatment with curcumin.A total of 53 419 438 clean sequence reads were generated from curcumin-treated mites and 52 286 859 clean sequence reads were generated from solvent-treated mites.Differentially expressed genes at 24 h and 48 h were 15450 and 15371,respectively.Meanwhile,significantly differentially expressed genes with the deviation probability value ?0.8 and fold change?2 were 111 and 96 at 24 h and 48 h,repsectively.In addition,the quantitative real-time PCR confirmed the accuracy of the results of RNA-seq and showed that the 15 genes of T.cinnabarinus were indeed involved in the response to curcumin.GO function enrichment analysis clarified the main biological processes that the significantly differentially genes involved in,which includs physiological processes,metabolism,single biological process,cell apoptosis,connection and catalysis,ion transport across the membrane,nerve conduction,etc.KEGG pathway enrichment analysis listed the top 20 biochemical metabolic pathways and signal transduction pathways,and endoplasmic reticulum protein processing and MAPK signal transduction pathway were the most representative biochemical pathways for both time ponts.In addition,23 genes were involved in the metabolism,signal transduction and phagocytosis of compounds,accounting for approximately 64% of the whole pathway genes.The genes contained in these GO terms and pathway terms were likely to be closely related to the mechanism of curcumin against mites.3.We obtained 23 significantly expressed genes related to metabolism and signal transduction,and determined the activity of glutathione-S-transferase to study detoxification metabolism of T.cinnabarinus.By using RPKM and Noiseq algorithm,we identified a total of 190 significantly differentially expressed genes from curcumin treated mites and 17 of them screened as common differentially expressed genes for both time points.Meanwhile,the gamma aminobutyric acid(GABA)as the target of some chemical agents and serine / threonine kinase as the target of curcumin in medicine were identified,as well as guanylate kinase,thioredoxin like 4A,Ras GTP enzyme associated with curcumin-targets.In the present study,we also obtained some enzyme genes related to calcium ions,including phospholipase C family,phospholipase A2,calmodulin,calnexin and endoplasmic reticulum stress related protein 1.Phospholipase A2 and calmodulin,as key genes of curcumin against T.cinnabarinus,were the most important signal transduction proteins and the most significantly differentially expressed genes.Moreover,we identified several GSTs genes and clarified the change of glutathione S-transferase with treating time to declare that the decrease of curcumin in mites was not caused by glutathione S-transferase.4.The c DNA sequences of calmodulin and phospholipase A2 in T.cinnabarinus were cloned and analyzed.We cloned and named two genes Tc Ca M and Tc PLA2,and submitted to NCBI with accession numbers KY436226 and KY436225,respectively.Based on the analysis of protein physicochemical properties,the predicted molecular weights of these two genes were 19.03 KDa and 12.04 KDa,and the isoelectric points were 4.32 and 4.97,respectively.By predicting the signal peptide,transmembrane protein structure,glycosylation sites and domain,we found that calmodulin and phospholipase A2 had no signal peptides and transmembrane structures.Calmodulin had four EF-hand domains and two glycosylation sites,and phospholipase A2 had only one glycosylation site without domain.The phylogenetic tree showed that the branch of the two genes are respectively corresponding to the two spotted spider mite gene is the closest to it and tetranychuscinnabarinus,two spotted spider mite close genetic relationship,the classification results with the results of the traditional sense of the same.The result of phylogenetic tree showed that the branches of these two genes were closest to those of Tetranychus urticae,which is consistent with the result of traditional classification.5.Tc Ca M and Tc PLA2 were expressed in all developmental stages of T.cinnabarinus and had important research value.The result of RT-q PCR showed that Tc Ca M and Tc PLA2 were expressed in all stages and the expression level in larvaes and nymphs were significantly higher than those in eggs and adults.The expression of calmodulin gene from high to low is as follows: nymph> larva> adult> egg.All stages had significance difference expect for nymph and larva.The expression of phospholipase A2 gene from high to low is as follows: larva> nymph> adult> egg.All stages had significance difference.The Tc Ca M and Tc PLA2 genes had become important proteins to maintain the normal life activities of the mites because of their special functions and existence in all different developmental stages.Therefore,the inhibition or activation of calmodulin and phospholipase A2 would have significant impact on the mites,and these two genes were of important value to the acaricide mechanism research.