林业科学2017，Vol.53Issue(6)：94-104,11.DOI:10.11707/j.1001-7488.201706011
转舞毒蛾LdCYP6AN15v1基因果蝇品系对氯虫苯甲酰胺胁迫响应
Responses of Transformant Drosophila Expressing LdCYP6AN15v1 Gene to Chlorantraniliprole Stress
摘要
Abstract
[Objective] Lymantria dispar is a major forest pest.Cytochrome P450 is ubiquitous key metabolic detoxification enzyme for xenobiotics in insects.This study on CYP6AN15v1 detoxifying pesticides aims to provide theoretical basis for L.dispar control.[Method] The full length cDNA of LdCYP6AN15v1 was cloned by RT-PCR technology.The transformant Drosophila vector expressing CYP6AN15v1 gene was constructed with the method of traditional restriction endonuclease digestion and ligation.Homozygous transformant Drosophila lines with LdCYP6AN15v1 were successfully constructed by using transformant technology.The effects of low dosage of chlorantraniliprole on cytochrome P450 activity and CYP6AN15v1 expression levels in transformant and untransformant Drosophila were examined using spectrophotometry and real-time RT-PCR technology,respectively.[Result] The full length cDNA of CYP6AN15v1 (namely LdCYP6AN15v1) was isolated from L.dispar transcriptome.The open reading frame (ORF) of LdCYP6AN15v1 was 1 539 bp encoding a protein of 512 amino acid residues with the molecular mass of 59.02 kDa.Phylogenetic analysis of CYP proteins showed CYP6AN15v1 of L.dispar clustered into a group with Spodoptera exigua and Helicoverpa armigera.The transformant Drosophila attP40>CYP6AN15v1 was detected 1 539 bp of target gene using DNA and cDNA as template showing successful expression of LdCYP6AN15v1 into transformant Drosophila.Compared to untransformant attP40 Drosophila,the susceptibility of attP40>CYP6AN15v1 Drosophila to chlorantraniliprole was significantly decreased by 2.92-fold of untransformant Drosophila for LC50.Under 7.17 mg·L-1 chlorantraniliprole stress,the cytochrome P450 activity and CYP6AN15v1 expression in L.dispar were dependent on time effects.The cytochrome P450 activity of attP40>CYP6AN15v1 Drosophila was from 1.09-to 1.93-fold of untransformant Drosophila while mRNA expression levels of CYP6AN15v1 in attP40>CYP6AN15v1 Drosophila were 44.54-to 137.80-fold of untransformant Drosophila showing induction effects.[Conclusion] The transformant Drosophila line attP40>CYP6AN15v1 was successfully constructed by using transgenic technology.The results suggest that the up-regulated expression of LdCYP6AN15v1 gene induced by chlorantraniliprole could enhance P450 activity in L.dispar larvae to detoxify the chlorantraniliprole.关键词
舞毒蛾/转基因果蝇/氯虫苯甲酰胺/CYP6AN15v1/诱导表达Key words
Lymantria dispar/transformant Drosophila/chlorantraniliprole/CYP6AN15v1/induced expression分类
农业科技引用本文复制引用
党英侨,殷晶晶,陈传佳,孙丽丽,刘鹏,曹传旺..转舞毒蛾LdCYP6AN15v1基因果蝇品系对氯虫苯甲酰胺胁迫响应[J].林业科学,2017,53(6):94-104,11.基金项目
国家自然科学基金项目(31570642) （31570642）
黑龙江省自然科学基金项目(C201409) （C201409）
东北林业大学本科生创新项目(201510225183). （201510225183）
