Molecular Cloning And Expression Analysis Of Halloween And Receptor Gene From Tetranychus Cinnabarinus(Boisduval)(Acari:Tetranychidae)

Ecdysone is an important hormone that regulates insect molting and metamorphosis.Its synthesis is mainly involved in the cytochrome P450 encoded by the Halloween gene,including CYP307A1(Spook,Spo),CYP306A1(Phantom,Phm),CYP302A1(Disembodied,Dib),CYP315A1(Shadow,Sad),CYP314A1(Shade,Shd),and work together with ecdysone receptors.Using ecdysone-regulated genes as targets,the application of RNAi to control pests is gradually becoming a research hotspot and provides a theoretical basis for the development of new pesticides.Tetranychus cinnabarinus is an important agricultural economic pests distributed worldwide.Its development process includes egg、larva、nymph、and adult,and molt at every stage of development.At present,the molecular mechanism of ecdysone synthesis of T.cinnabarinus is unclear;This article takes T.cinnabarinus as a research object,the Halloween gene and nuclear receptor gene of T.cinnabarinus were cloned by RT-PCR and RACE,The sequence characteristics were analyzed by bioinformatics;on this basis,RT-qPCR was used to study the mRNA expression patterns of these genes in different stages of the development of T.cinnabarinus;The toxicity of ecdysone agonist chromafenozide to T.cinnabarinus was determined,The mRNA expression levels of Halloween gene and nuclear receptor gene of T.cinnabarinus treated with chromafenozide were studied.The results are as follows:1.Bioinformatics analysis of Halloween gene and nuclear receptor gene of T.cinnabarinusThree Halloween genes and one nuclear receptor gene of T.cinnabarinus were cloned for the first time,and named TcDib,TcSad,TcShd,TcEcR,respectively.TcDib(GenBank accession number:MN011166)has a total length of 2201bp,including 1560bp open reading frame(ORF),519aa be coded,59.315 kDa molecular weight and 8.9 theoretical PI.TcSad(GenBank accession number is MN807301),including 1965bp open reading frame(ORF),654 aa be coded,74.58 kDa molecular weight and 8.69 theoretical PI.TcShd(GenBank accession number:MN807300)has a total length of 2354bp,including 1446bp open reading frame(ORF),481aa be coded,54.68kDa molecular weight and 9.0 theoretical PI.TcEcR(GenBank accession number:MN011167)has a total length of 3937bp,including 1650bp open reading frame(ORF),549aa be coded,61.91 kDa molecular weight and 7.7 theoretical PI.The conserved domains of these genes were analyzed:The three Halloween genes TcDib,TcSad and TcShd of T.cinnabarinus all have typical P450 domains WxxR(Helix-C),P/IPGPxG/PxP(PERF motif),ExxR(Helix-K),A/GGxE/DTT/S(Helix-I)and heme binding domain PFxxGxRxCxG/A;The nuclear receptor gene TcEcR has a typical DNA binding domain(DBD)and ligand binding domain(LBD).After homology analysis,it was found that these genes were highly conserved with the related sequences of this gene in other species;The phylogenetic study based on amino acid sequence showed that the amino acid sequence encoded by Halloween gene and receptor gene of T.cinnabarinus was clustered together with corresponding homologous sequences in other insects and mites,all of them have the closest relationship with Tetranychus urticae and Panonychus citri.2.Development-specific expression patterns of the Halloween gene and nuclear receptor gene in T.cinnabarinusIn this paper,the mRNA expression patterns of Halloween gene and nuclear receptor gene in different stages of the development(egg、larva、nymph、and adult)of T.cinnabarinus were studied by real-time fluorescence quantitative technique.The experimental results show that these genes are expressed in each stage of development,but the expression level is different,The expression levels of TcDib,TcSad and TcShd of Halloween gene were the highest expression on the second day of protonymph,the female adult and the second day of deutonymph respectively,The expression of TcEcR and TcRXRl was the highest on the second day of deutonymph and TcRXR2 was the highest on the second day of protonymph;The expression of these genes increased before molting,but decreased after molting.It is speculated that these genes may be related to molting of T.cinnabarinus.In addition,it was found that the expression of these genes in female adult mites was generally higher than that in male adult mites,indicating that ecdysone may also be related to female reproduction.3.The toxicity of chromafenozide to T.cinnabarinus and its effect on the mRNA expression of Halloween gene and nuclear receptor geneIn this paper,different concentrations of chromafenozide solutions were used to bioassay the larvae of T.cinnabarinus,and the LC50 value was 52.75 mg/L.Then treated with different concentrations(LC10,LC30,LC50,LC70)of the larvae of T.cinnabarinus,their ecdysisrate,mortality,molting duration were analyzed and the content of ecdysone in the body was determined using test kits.The results showed that the ecdysisrate,mortality,molting duration and ecdysteroid content of T.cinnabarinus could change with the concentration of insecticide.The ecdysisrate increased with the increase of drug concentration;The mortality increased with the increase of drug concentration,the LC70 treatment group increased by 65.6%compared with the control group;the molting duration was shortened with the increase of drug concentration,the LC70 treatment group was 0.8 times that of the control group;The content of ecdysone in the body of the larvae of T.cinnabarinus was increased with the increase of drug concentration,and the LC70 treatment group was 1.62 times that of the control group.The results showed that the ecdysis process of T.cinnabarinus was affected by chromafenozide.At the molecular level,RT-qPCR was used to detect the mRNA expression levels of the Halloween gene(TcDib,TcSad,TcShd)and nuclear receptor genes(TcEcR,TcRXR1,TcRXR2)after drug treatment.The results showed that the expression levels of Halloween genes involved in ecdysone synthesis were increased after treatment with diferent concentrations of chromafenozide.The mRNA expression of TcDib in the LC30 treatment group was 1.76 times compared with the control group;The mRNA expression of TcSad in the LC10 treatment group was 1.54 times compared with the control group;The mRNA expression of TcShd in the LC70 treatment group was 1.77 times compared with the control group.The expression of nuclear receptor gene was increased after treatment with different concentrations of chromafenozide.The mRNA expression of TcEcR in the LC30 treatment group was 2.69 times compared with the control group;The mRNA expression levels of TcRXR1 and TcRXR2 in the LC50 treatment group were 3.24 and 3.02 times compared with the control group.It is suggested that chromafenozide can induce and regulate the transcription of Halloween gene and nuclear receptor gene of T.cinnabarinus.In conclusion,the sequence characteristics of Halloween gene and nuclear receptor gene of Tcinnabarinus were analyzed and their mRNA expression patterns were discussed,laid the foundation for the subsequent analysis of the function of these genes,it also provides a theoretical basis for using these genes as targets to prevent and control T.cinnabarinus.