关键词:  梨小食心虫, 中肠转录组, 基因表达, 消化酶, 解毒酶

Abstract: 【Aim】 This study aims to explore the digestive and detoxification enzyme genes highly expressed in the midgut of Grapholita molesta larvae so as to provide the theoretical basis for the future research of novel pesticides and transgenic crops targeting intestine. 【Methods】 Based on the FPKM values of high-throughput transcriptome sequencing data of the 4th instar larval midgut of G. molesta, the highly expressed genes were screened and subjected to GO function annotation and KEGG pathway enrichment analysis, and the highly expressed genes of the digestive and detoxification enzymes were screened by alignment using BLAST software. Phylogenetic analysis of these highly expressed digestive and detoxification enzymes and the homologous proteins from other lepidopteran insects was performed using MEGA software. Quantitative analysis and verification of the expression levels of the representative digestive and detoxification enzyme genes highly expressed in the midgut of G. molesta larvae at different instars were carried out by qRT-PCR. 【Results】 A total of 103 677 genes highly expressed in the migdut of the 4th instar larvae of G. molesta were annotated in GO database, including 41 branches of three function categories cellular component, molecular function and biological process. KEGG pathway analysis showed that 10 846 highly expressed genes are involved in five types of biochemical metabolism pathways. Seventeen digestive enzyme genes ［five trypsin (TRY) genes, three aminopeptidase (APN) genes and nine carboxypeptidase (CP) genes］ and 32 detoxification enzyme genes ［11 glutathione Stransferase (GST) genes, 13 cytochrome P450 (CYP450) genes and eight carboxylesterase (CarE) genes］ with complete open reading frames were obtained. The results of phylogenetic analysis indicated that the homologous clustering branches of the digestive enzymes of G. molesta were scattered, and the homologous clustering branches of the GST and CYP450 proteins of G. molesta were concentrated, but they were all clustered with the homologous proteins of at least one lepidopteran insect. qRT-PCR verification result showed that the expression levels of digestive enzyme and detoxification enzyme genes in the midgut of G. molesta larvae at different instars were significantly different, with the highest expression levels in the 4th instar larval stage. 【Conclusion】 In this study, we successfully screened and verified some digestive enzyme and detoxification enzyme genes highly expressed in the larval midgut of G. molesta, and clarified their evolutionary relationship with homologous proteins of other lepidopteran insects. These results provide references for the transcriptome analysis of other related species of Lepidoptera and the study of insect pest control targeting intestine.

Key words:  Grapholita molesta, midgut transcriptome, gene expression, digestive enzyme, detoxification enzyme