关键词: 苹果蠹蛾, 细胞色素P450, 植物次生物质, 基因表达, 解毒代谢

Abstract: 【Aim】 This study aims to clone the sequences of two cytochrome P450 genes CYP332A19 and CYP337B19 from the codling moth, Cydia pomonella, and to analyze their structure properties and expression profiles, so as to further understand the roles of the two genes in the detoxification of plant secondary substances and to provide evidence for further functional research. 【Methods】 The cytochrome P450 gene cDNA sequences were searched from the transcriptome database of C. pomonella by local BLSTA, and the coding regions of the target genes were cloned using RT-PCR. Bioinformatics software was used to analyze the sequence characteristics and phylogenetic relationship of the target genes with P450 genes of other related species. RT-qPCR was employed to determine the expression patterns of the CYP450 genes in different developmental stages (egg, 1st-5th instar larva, pupa, and adult), various tissues (head, epidermis, fat body, midgut, and Malpighian tubules) of the 4th instar larvae, and the 4th instar larvae of C. pomonella fed with artificial diets supplemented with 0.1% coumarin and 0.5% quercetin, respectively, for 2 d. 【Results】 The full-length cDNA sequences of CYP450 genes CYP332A19 (GenBank accession no.: MF574708) and CYP337B19 (GenBank accession no.: MF574697) were cloned, with an open reading frame (ORF) of 1 518 bp and 1 491 bp in length, respectively, encoding 505 and 496 amino acids, respectively. The molecular mass of CYP332A19 and CYP337B19 is 58.586 and 57.734 kD, respectively, and their theoretical isoelectric points are 8.99 and 7.61, respectively. Domain analysis showed that both CYP332A19 and CYP337B19 contain five conserved regions of CYP450 genes, including the heme binding domain. Phylogenetic analysis indicated that CYP332A19 and other CYP332A genes such as CYP332A9 from Epighyas postvittana were clustered together, while CYP337B19 and CYP337B genes such as CYP337B12 from Cnaphalocrocis medinalis and CYP337B11 from Zygaena filipendulae were gathered in another branch. The RT-qPCR results showed that the mRNA levels of CYP332A19 and CYP337B19 in the larval stage of C. pomonella were higher than those in the egg stage. Moreover, CYP332A19 and CYP337B19 had the highest expression levels in the fat body and midgut of the 4th instar larva, respectively. The relative expression levels of CYP332A19 and CYP337B19 in the 4th instar larvae fed on the artificial diets containing 0.1% coumarin and 0.5% quercetin, respectively, for 2 d were significantly higher than those in the control group (fed on the artificial diet containing 2% DMSO). 【Conclusion】 CYP332A19 and CYP337B19 show the highest expression level in the fat body and midgut of C. pomonella larva, respectively, and are upregulated in the larvae fed with the artificial diets containing coumarin and quercetin, suggesting that the two genes might play a critical role in the detoxification of xenobiotics. The results of this study are helpful for understanding the detoxification mechanism to plant secondary metabolites in the codling moth and provide new ideas for the control of the moth.

Key words: Cydia pomonella; cytochrome P450, plant secondary substance, gene expression, detoxification metabolism