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

【目的】舞毒蛾是林业重要害虫,细胞色素P450是昆虫体内广泛分布的参与外源化合物代谢关键酶系,探讨P450家族基因CYP6AN15v1对杀虫剂代谢解毒功能,为舞毒蛾有效治理提供依据。【方法】通过RT-PCR法获得Ld CYP6AN15v1基因c DNA全长,采用传统酶切连接的方法构建转CYP6AN15v1基因果蝇载体,通过转基因技术获得表达Ld CYP6AN15v1果蝇品系(命名为att P40>CYP6AN15v1)。采用分光光度计法研究低剂量氯虫苯甲酰胺(7.17mg·L^(-1))处理对转基因和非转基因果蝇品系细胞色素P450活性的影响,并采用qRT-PCR法测定其对CYP6AN15v1基因表达的影响。【结果】从舞毒蛾无参照转录本文库中克隆获得CYP6AN15v1全长基因,编码512个氨基酸,蛋白分子质量为59.02 k Da;系统进化树分析表明CYP6AN15v1与甜菜夜蛾和棉铃虫关系较近。以DNA和c DNA为模板,att P40>CYP6AN15v1果蝇品系均检测到1 539 bp目的基因,表明Ld CYP6AN15v1基因成功整合到果蝇基因组。与非转基因att P40果蝇品系相比,转基因att P40>CYP6AN15v1果蝇品系对氯虫苯甲酰胺的敏感性显著降低,致死中浓度LC50为非转基因果蝇的2.92倍;低剂量(7.17 mg·L^(-1))氯虫苯甲酰胺胁迫下,舞毒蛾细胞色素P450酶活性和CYP6AN15v1基因的诱导作用呈现时间效应,att P40>CYP6AN15v1果蝇品系P450活性为非转基因果蝇的1.09~1.93倍,主要表现为诱导效应;att P40>CYP6AN15v1果蝇品系的CYP6AN15v1基因mRNA表达量呈诱导激活,其表达量为非转基因的44.54~137.80倍。【结论】利用转基因技术成功构建了转Ld CYP6AN15v1果蝇品系att P40>CYP6AN15v1;氯虫苯甲酰胺可能通过诱导Ld CYP6AN15v1基因mRNA的上调表达而增强黑腹果蝇P450酶活性,从而参与对氯虫苯甲酰胺的解毒作用。

【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 c DNA of Ld CYP6AN15v1 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 Ld CYP6AN15v1 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 c DNA of CYP6AN15v1（ namely Ld CYP6AN15v1） was isolated from L. dispar transcriptome. The open reading frame（ ORF） of Ld CYP6AN15v1 was 1 539 bp encoding a protein of 512 amino acid residues with the molecular mass of 59. 02 k Da. Phylogenetic analysis of CYP proteins showed CYP6AN15v1 of L. dispar clustered into a group with Spodoptera exigua and Helicoverpa armigera. The transformant Drosophila att P40〉 CYP6AN15v1 was detected 1 539 bp of target gene using DNA and c DNAas template showing successful expression of Ld CYP6AN15v1 into transformant Drosophila. Compared to untransformant att P40 Drosophila,the susceptibility of att P40 〉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 att P40 〉CYP6AN15v1 Drosophila was from 1. 09-to 1. 93-fold of untransformant Drosophila while mRNA expression levels of CYP6AN15v1 in att P40 〉CYP6AN15v1 Drosophila were 44. 54-to 137. 80-fold of untransformant Drosophila showing induction effects. 【Conclusion】The transformant Drosophila line att P40 CYP6AN15v1 was successfully constructed by using transgenic technology. The results suggest that the up-regulated expression of Ld CYP6AN15v1 gene induced by chlorantraniliprole could enhance P450 activity in L. dispar larvae to detoxify the chlorantraniliprole.