| Topic Basis: As the country with the richest Chinese medicine resources in the world,China’s herbal medicine industry is also on a trend of increasing year by year.The quality of Chinese medicinal materials determines the rise and fall of the Chinese medicine industry.However,Chinese medicinal materials are inevitably damaged by pests in the process of production,transportation and storage.Therefore,the effective prevention and control of insect pests is the key factor to solve the problem of good quality of Chinese medicinal materials.Coleoptera insects are the dominant species of Chinese medicine storage pests,in which,Tribolium castaneum is the first one to complete the whole genome sequencing.Cytochrome P450 enzymes are one of the three major detoxification enzymes in insects.The increase of their content and activity is also the main reason for the development of insecticides resistance in insects.In the previous studies,our research group has found that the injection of ds Tc Cy303a1 chould result in 100% mortality during the larvae and pupae stage of T.castaneum.However,the specific biological function of this gene is still unclear.Therefore,in this study,the RNAi technology and UHPLC-Q-TOF-MS/MS metabolomics technology were combined to in-depth analyze the biological functions of Tc Cy303a1 gene.Objective: Based on the T.castaneum genome database,the c DNA sequence of TcCyp303a1 gene was obtained,and the m RNA&protein expression characteristics of the target gene TcCyp303a1 were analyzed.The biological function of the target gene TcCyp303a1 was preliminarily analyzed by combining RNAi technology with phenotypic observation.The important metabolic pathways related to TcCyp303a1 were explored by metabolomics technology and enrichment analysis of metabolic pathways,and the potential biological functions of TcCyp303a1 were analyzed by nontarget metabolomics and molecular biology technology.Methods: 1.Based on the T.castaneum genome database,the TcCyp303a1 gene was searched,analyzed and cloned,and the phylogenetic tree of the gene was constructed.RT-q PCR technology was used to analyze the m RNA expression levels in different developmental time and different tissue and body parts of TcCyp303a1.The peptide fragment of TcCyp303a1 was used to prepare antibodies and the expression levels of TcCyp303a1 at different development times were analyzed by western blot.2.The expression of TcCyp303a1 gene was silenced by RNAi technique.the gene silencing efficiency,protein changes after silencing and the observation of the phenotype were conducted to to investigate the biological function of TcCyp303a1 in vivo.The immunohistochemical technique was used to analyze the tissue distribution of TcCyp303a1 protein after the depression of TcCyp303a1 gene.3.At the 24 h after the depression of the TcCyp303a1 gene,the changes of metabolites in T.castaneum were analyzed by using UHPLC-Q TOF-MS/MS non-targeted metabolomics technology and multivariate statistical method,and the biological function of TcCyp303a1 was further explored by combining molecular biology technology.Results: 1.Based on the genomic database of T.castaneum,the sequence of TcCyp303a1 were searched,then the gene structure was analyzed.The amino acid sequence showed that TcCyp303a1 contented 494 amino acids and the molecular weight was 56 k Da.The results of RT-q PCR showed that the TcCyp303a1 was highly expressed in the late eggs,early larvae,the fifth day of the 6th instar larvae and the fourth day of the pupal stage,also highly in the thoracic epidermis,abdominal epidermis,gut and fatbody tissue parts of T.castaneum.Western Blot showed that TcCyp303a1 protein was highly expressed mainly in the fifth day of 6th instar larvae,the fifth day of the pupal stage and the first day of the adult,which was basically consistent with the situation of m RNA expression.2.The ds RNAs of the target gene TcCyp303a1 and the Tc Vermilion were injected into the insect coelom at the second day of 5th,6th and pupal stage of T.castaneum,respectively.The results of gene detection and phenotypic observation showed that the gene silencing efficiency were 79%,86% and 93% in the 5th,6th and pupal stages after the injection of ds TcCyp303a1,respectively.Moreover,the insects suffered from molting failure and died after the depression of TcCyp303a1 at each stage.The results of western blot showed that protein levels of TcCyp303a1 were significantly decreased after the injection of ds TcCyp303a1 during the 5th,6th and pupal stages.The immunohistochemical experiments showed that the content of TcCyp303a1 in the thoracic epidermis was significantly reduced after the injection of ds TcCyp303a1.3.The results of UHPLC-Q TOF-MS/MS analysis showed that TcCyp303a1 regulated the metabolic profile of the sixth-instar larvae of T.castaneum.The multivariate statistical analysis showed that a total of 41 differential metabolites were identified,and two most important metabolic pathways,the biosynthesis of unsaturated fatty acids and the metabolism of tryptophan,were screened out through pathway enrichment analysis.RT-q PCR showed that the gene expressions of FAS and ELO were significantly decreased.The significantly decreased contents of melatonin and 5-HT were also detected by using Elisa after the depression of TcCyp303a1.Conclusion: c DNA sequence of TcCyp303a1 was cloned in this study.To clarify the molecular characteristics of m RNA and protein of TcCyp303a1 gene of T.castaneum.The biological functions of the TcCyp303a1 gene were explored based on RNAi technology and UHPLC-Q-TOF-MS/MS metabolomics technology.The results of metabolomics experiments indicated that the depression of TcCyp303a1 significantly reduced the tryptophan metabolism pathway and the biosynthesis pathway of unsaturated fatty acids in T.castaneum.The results will promote the application of TcCyp303a1 in the field of pest control. |
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