Analysis Of Ecdysteroid Biosynthesis And Signaling Pathway In Panonychus Citri(McGregor)(Acari:Tetranychidae)

The citrus red mite,Panonychus citri(McGregor)is an important agricultural pest that causes serious damage in orange orchards and affects the yield and quality of citrus.Currently,the control of P.citri is still dominated by chemical acaricide.However,due to the short developmental duration of the mite,the overlapping of generations,the rapid propagation rate,and the unreasonable use of field acaricides,resistance problems of mites are becoming more and more serious.Thus it is urgent to find new targets and to develop new acaricides for the prevention and control of P.citri.In recent years,the development of new insecticide with molecular targets related to the growth and development of pests has become a popular research topic.Ecdysone is an important hormone regulating the growth and development of insects.The phytosterols(animal cholesterol)obtained by insects are catalyzed by a series of enzymes encoded by the Halloween gene to bioactive 20 E,which is binded to the heterodimer of the ecdysone receptor and the super-valve protein USP.Then this complex initiates a series of early and late cascades of 20 E signaling pathway to affect cell proliferation,differentiation and programmed cell death and to regulate the whole process of development and molting.So far,the research on ecdysteroid of P.citri are rare,and there is no report on the ecdysteroid synthesis and signaling mechanism of P.citri.This dissertation is based on ecdysteroid synthesis and signal transduction pathway of P.citri.Based on comprehensively excavate the information of ecdysone synthesis and signaling pathway homologous gene information,the key genes involved in molting process of different development stages were screened,and the expression patterns of these genes in step-in and step-out the molting of the different development stages were analyzed,where the function of key genes in the molting process was verified.Morever,the active ecdysteroid was identified,and the tissues of ecdysteroid biosynthesis and signaling transmission were localized.Finally,the binding mode of the ecdysone receptor complex EcR-RXR1/RXR2 was analyzed.The results have important theoretical and temporal value for revealing the molecular regulation mechanism of P.citri ecdysone biosynthesis and signaling pathway as well as exploring new targets and developing new acaricides based on the molecular regulation mechanism.The main results are as follows: 1 Exploration of the related genes in regulating molting of P.citriHigh-throughput sequencing technology was used for sequencing the samples of three molting process from larva to adult of P.citri.It was found that there were 4,456 differential expressed genes in the three times of molting.GO cluster showed that most of the differential genes were involved in hormone synthesis,protein synthesis and absorption and nutrient synthesis and transportation.The KEGG pathway analysis showed that the ecdysone pathway genes,chitin related genes,cuticle protein genes and ABC transporter genes had the highest enrichment ratio.The comparative analysis of the differential genes in the three molting process found that there was no common differentially expressed gene in the molting process between the process of larva to protonymph and the process of the protonymph to the deutonymph.The molting process from deutonymph to adult has 50% of the same differentially expressed genes with the process from protonymph to deutonymph,suggesting there is a difference in the molecular regulation mechanism of the molting process between larva to protonymph and protonymph to deutonymph.Comparative analysis of the transcriptome of three molting process,the high expression genes from larva to protonymph was opposite to express low aboundance in the molting process from protonymph to deutonymph;on the contrary,there were a same result between the highly expressed genes from protonymph to deutonymph and the lower expressed genes from larva to protonymph,suggesting that there is a difference in the temporal dynamic of gene expression in the molecular regulation of the molting process.The above results laid a molecular basis for the study of the regulation and development network of P.citri.2 Identification of key genes in ecdysteroid biosynthesis and signaling pathway 2.1 Identification of ecdysone synthesis and signaling pathway homologous genes based on the genome and transcriptome of P.citriBased on a combination of genome and transcriptome from different developmental stages of P.citri,a total of 15 full-length cDNA sequences of ecdysteroid biosynthesis and signaling pathway homologous genes were obtained,including 5 ecdysone synthase genes PcSro,PcSpo,PcDib,PcSad and PcShd,of which four P450 genes have conserved domains: WxxxR(Helix-C),GxR/DTT/S(Helix-I),ExxR(Helix-K),PxxFXPE/DRF(PERF motif),and hemoglobin binding site PFxxGxRxCxG/A;8 ecdysone signaling pathway nuclear receptor genes PcEcR,PcRXR1,PcRXR2,PcE75,PcE78,PcHR3,PcHR38 and PcFTZ-F1,with typical nuclear receptor conserved domains: DNA binding domain,ligand Binding domain and 2 zinc finger structures in the C domain;and 2 transcription factors PcE74 and PcKr-h1.2.2 Expression patterns of ecdysteroid synthesis and signaling pathway genes in step-in and step-out moltingThe expression patterns of 14 genes of ecdysteroid biosynthesis and signaling pathway in 8 h step-in and step-out molting were analyzed by qRT-PCR.The results showed that the expressions of PcSpo,PcDib and PcRXR2 were increased at 8 h step out each molting and decreased at 8 h step in the molting.The expression of PcRXR1,PcE78 and PcFTZ-F1 were decreased at 8 h step out molting,and increased at 8 h step in the molting.The above genes exhibited two different "Zigzag-like" expression patterns in different developmental stages of P.citri.The expression levels of PcSad and PcHR38 in the three stages of protonymph,deutonymph and adult were increased at 8 h after the molting,and in the end of the egg and 8 h step out molting of larva,protonymph and deutonymph,the expression was reduced.The expression level at the end of egg stage was higher than that at 8 h after the molting of larva.The expression level of PcEcR was highly expressed before and after each molting,but the difference was not significant.PcShd and PcKR-H1 were highly expressed in the adult stage,and there was no significant difference in the expression levels of eggs,larva,protonymph and deutonymph,and remained at a lower level.It is suggested that these genes are involved in the development of P.citri and play different roles at different development stages.3 Ponasterone A is the active ecdysteroid of P.citri 3.1 The effect of ecdysteroid biosynthesis gene PcSpo on the molting processThe dsRNA of the ecdysteroid biosynthesis gene PcSpo was fed to the protonymph 8 h step out molting.It was found that the expression of PcSpo was significantly down-regulated after feeding with dsRNA for 24 h,and the silencing efficiency reached 56%.The growth and development time was delayed,and the rate of molting was decreased with the phenotype that protonymph mites cannot be normally molted and trapped in the old epidermis,and a cumulative mortality rate was 35%.At the same time,qRT-PCR was used to detect the changes of ecdysone pathway-related genes after interference with PcSpo.The results showed that after PcSpo was effectively silenced,and the expressions of PcSib,PcSad,PcRXR2,PcE74,PcHR38 and PcKR-H1 were reduced,which had a same expression pattern with PcSpo.The expression levels of PcShd,PcRXR1,PcE78 and PcHR3 were up-regulated in the opposite expression patterns of PcSpo;the expression level of PcE75 was also significantly increased.The above results indicate that PcSpo is involved in the molting process of P.citri whole claws.3.2 Effect of Ponasterone A on the molting process of P.citriAfter interfering with PcSpo expression,the active ecdysteroid hormone of P.citri was identified by hormone rescue method.The results showed that after feeding with ds PcSpo and 20 E mixture for 36 h,the molting rate of mite was 26.6%,which was basically consistent with the rate of 25.7% of dsPcSpo feeding.It indicated that 20 E could not rescue the inhibitory effect of ds PcSpo on the molting;After feeding dsPcSpo and Ponasterone A for 36 h,the molting rate was 90%,indicating that Ponasterone A can rescue inhibiting effect of dsPcSpo on molting.The above results confirmed that Ponasterone A is an active ecdysteroid hormone of P.citri.4 The tissue of ecdysteroid biosynthesis and signaling tranmissionThe in situ hybridization technique was used to locate the expression site of the ecdysteroid biosynthesis pathway gene PcSpo and the signal pathway gene PcRXR2.The results showed that the ecdysteroid biosynthesis genes PcSpo has a signal in the anterior region of the center nervous mass and the signal of nuclear receptor PcRXR2 is in the center nervous mass and its surrounding lipid globule of P.citri,indicating that the central nervous system tissue is the main tissue of P.citri ecdysteroid biosynthesis and signal transduction.5 Analysis of the binding mode of ecdysone receptor heterodimer EcR-RXR1/RXR2 of P.citri 5.1 RNAi effect of EcR/RXR1/RXR2 on the molting of P.citriWe analyzed the effect of RNAi on depletion of ecdysone receptor EcR-RXR1/RXR2 during the molting of P.citri.The deutonymph were fed with dsPcEcR,ds PcRXR1,ds PcRXR2 and dsPcRXR1 + dsPcRXR2 post 24 h,and silencing efficiencies of PcEcR,PcRXR1 and PcRXR2 were respectively 52%,69.8% and 40.7%.After 72 h,96% of mites in control group had molted to the adult mites,whereas mites feeding with dsPcEcR,dsPcRXR1,dsPcRXR2 and ds PcRXR1 + dsPcRXR2,showed the molting rates was 69%,46.34%,51.51% and 35%,respectively.There were phenotypes that could not normally enter the molting state,and the mortality rates were 23%,48.78%,45.45% and 55.0%,respectively.It is suggested that both the ecdysone receptor EcR and the nuclear receptor RXR1/RXR2 are involved in the ecdysone signaling process.5.2 Analysis of the expression pattern of EcR-RXR1/RXR2 at different time pointsThe expression pattern of ecdysone receptor heterodimer genes PcEcR-RXR1/RXR2 at different developmental time points was analyzed by qRT-PCR.The results showed that the expression of PcEcR gradually increased from 4 h to 24 h after the molting,and gradually decreased,reaching the lowest level at 36 h among all time points.The expression level of PcRXR2 increased from 16 h to 24 h,reaching the highest at 24 h among all time points,then decreased gradually and reaching the lowest level at 36 h among all time points.The expression level of PcRXR1 was not significantly different from 4 h to 16 h after molting,and then began to increase from 16 h to 28 h.The expression reached the highest level at 28 h among all time points.Looking at the development of P.citri,the mites continued to ingest food from 4 h to 24 h with the increased body size and the increased activity capacity.After 28 h,the activity capacity was decreased and began to enter the molting stage.After 32 h,the remained the molting state was completed.Combined these genes expression pattern and the developmental process of the mites,it is suggested that PcEcR may form a heterodimer with PcRXR2 binding to ecdysone to form a complex,which induces the expression of genes downstream of the ecdysone signaling pathway to regulate the development process,however,PcEcR and PcRXR1 may form a heterodimer to trigger the molting process of mites.5.3 Transcriptome analysis of different developmental time points in deutonmymph of P.citriBased on the binding model of ecdysone receptor heterodimer EcR-RXR1/RXR2 at different developmental time points,high-throughput sequencing technology was used to identify different developmental time points(8 h,24 h and 36 h).The results showed that there were 1474 differential expressed genes in comparison among 8 h,24 h and 36 h.Among them,there were 490 differential genes,including 316 up-regulated genes and 174 down-regulated genes in 8 h vs 24 h,and 984 differential genes,including 607 up-regulated genes and 377 genes were down-regulated in 24 h vs 36 h.The genes of cuticle protein and ABC transporter were up-regulated in comparison among 8 h,24 h and 36 h,suggesting that the cuticle protein was continuously biosynthesized during the development of P.citri to increase the fimness of the epidermis and improve its survival rate.The specific highly expressed genes including rhodopsin,cytochrome b and ABCH5 in 8 h vs 24 h suggest that PcEcR formed a heterodimer with PcRXR2 during the development from 8 h to 24 h,and then regulates the body color change and eye formation process of P.citri.The specific high expression genes in 24 h vs 36 h were the chitinase gene and cuticle protein CPAP2 suggesting that PcEcR formed a heterodimer with PcRXR1 to regulate chitin degradation and new epidermis formation.