Analysis Of Transcriptome Data And Function Of Cuticle Protein Gene CPAP3-D2 From Tetranychus Cinnabarinus

Tetranychus cinnabarinus is a destructive phytophagous pest.So far,the main method to control it is still the application of chemical insecticide.However,the overuse of chemicals has been inevitably leaded to insect resistance,making its control extremely difficult.Studies have shown that the main components of insect epidermis are chitin and cuticle protein,which interact with each other to form a stable structure to support and protect the insect body,play an important role in the physiological processes of insect growth,reproduction and environmental adaptation.A large number of studies in the domestic and overseas have focused on the synthesis and degradation metabolism of insect chitin and cuticle proteins as pest control targets to destroy the integrity of the epidermal composition and structure,and provide ideas for pest control.In this study,we conducted a high-throughput RNA-seq technology to identify the variations in transcriptomic profile of T.cinnabarinus larvae which were exposed to different concentrations of diflubenzuron.Meanwhlie,the full-length cDNA of cuticle protein gene CPAP3-D2 from T.cinnabarinus was cloned and gene expression and function were studied.The main results were as follows:（1）In the transcriptome analysis of T.cinnabarinus.Compared with the control,470 genes were differentially expressed（DEGs）in LC₅₀-treated group,157 and 313 genes expression level were up-regulated and down-regulated,respectively.Moreover,49 genes were differentially regulated in LC₇₀-treated group（4 up-regulated and 45 down-regulated）.In addition,151 and 3genes were specifically expressed in LC₅₀-and LC₇₀-treated groups,respectively,and they shareed41 DEGs.The results of GO enrichment showed that DEGs were mainly enriched in the processes of"ion transmembrane transport","virus particle","extracellular region","lipid transporter activity"and"oxidoreductase activity"after the treatment with low concentration of diflubenzuron.Similarly,in LC₇₀-treated group,"energy coupled proton transport","metal ion binding","lipid particle"were the most enriched items in the categories.The results of DEGs enrichment pathways demonstrated that the most enriched pathways included"insect hormone biosynthesis"and"lysosome"in LC₅₀-treated group.In LC₇₀-treated group,"insect hormone biosynthesis","lysosome"and"oxidative phosphorylation"were the most enriched pathway.We also The detoxification enzymes involved in the transcritome of T.cinnabarinus were identified and analyzed in this study,and the results suggested that they were 34 cytochrome P450 genes,17GST genes,12 ACh E and 9 ABC transporter genes.In addition,differentially expressed genes analysis showed that the gene expression levels of detoxification enzymes were generally enhanced.At the same time,7 and 11 genes were involved in chitin synthesis and degradation ways,respectively.The expression level of the most genes involved in chitin synthesis and degradation pathway were generally up-regulated after exposure to different concentrations of diflubenzuron.Among them,compared with the control group,the expressions of TcGFAT2,TcGNA and TcPGM genes in the chitin synthesis pathway were up-regulated in LC₅₀-treated group,while the expressions of TcHEX1,TcHEX2,TcG6PI2,TcGFAT2 and TcUAP genes were up-regulated in LC₇₀-treated group.Meanwhile,in the chitin degradation pathway,the expression of three chitinase genes（TcCh T2,TcCHT4,TcCHT8）were up-regulated after exposed to different concentrations of diflubenzuron.（2）The full-length cDNA sequence of cuticle protein CPAP3-D2 gene from T.cinnabarinus was 990 bp,encoding 271 amino acids with the predicted molecular weight of 29.67 KDa and p I of 6.04.TcCPAP3-D2 has a typical N-terminal signal peptide of the CPAP3 cuticle protein family and three consecutive conserved domains of ChtBD2 without transmembrane structure.The results of multiple sequence alignments showed that TcCPAP3-D2 has the highest amino acid sequence identify（43.54%）with CPAP3-D2 from Tribolium castaneum,and the lowest amino acid sequence identify from Acyrthosiphon pisum（27.99%）.Phylogenetic results showed that CPAP3-D2 gene of T.cinnarinus was clustered into a branch of CPAP3-D2 gene of other insect species,and the phylogenetic relationship was as follows:（（（（（Aedes aegypti+Culex quinquefasciatus）+Acyrthosiphon pisum）+Pediculus humanus corporis）+（Tribolium castaneum+Nasonia vitripennis））+Tetranychus cinnabarinus）.RT-qPCR was used to detect the expression level of TcCPAP3-D2 during all the developmental stages.The results revealed that the expression level of TcCPAP3-D2 gene was expressed throughout different developmental stages,and the highest expression was shown at the stage of nymph.Its expression pattern was consistent with ecdysis,that is,the expression reached the highest level before each ecdysis and decreased rapidly after each ecdysis.Finally,RNA interference experiments showed that the mortality rate of T.cinnabarinus reached 80%after feeding dsTcCPAP3-D2 solution to T.cinnabarinus nymph at 48h.The results of RT-qPCR analysis showed that the gene expression of CPAP3-D2 was significantly down-regulated in the RNA silence group,compared with the control group（P<0.05）,which was about 1/5 of that in the control group,indicating that TcCPAP3-D2 gene may play an important role in the growth and development of T.cinnabarinus nymph.In summary,from the perspective of chitin metabolism,the second-generation transcriptome sequencing technology was used to explore the responding mechanism of T.cinnabarinus to DFB exposure.At the same time,the sequence characteristics and gene expression of CPAP3-D2 from T.cinnabarinus were analyzed,and the biological function of CPAP3-D2 gene was explored using RNA interference technology.All the results in the present study were focused on the epidermal metabolism of T.cinnabarinus,and they will provide new strategies and ideas for the prevention and control of T.cinnabarinus.