转飞蝗CYP408B1和CYP409A1基因果蝇品系对杀虫剂的敏感性

摘要

Objective]Cytochrome P450s are ubiquitous metabolic detoxification enzymes in insects.Locusta migratoria is a major agricultural pest. It is important to identify and validate the metabolic detoxification function of P450. The objectives of this study are to construct transgenicDrosophilamelanogaster lines with locustCYP408B1 andCYP409A1genes, determine the susceptibility of transgenic Drosophila expressed exogenousCYP408B1 orCYP409A1 to insecticides, and to reveal the characteristics of locustCYP408B1 andCYP409A1.[Method] Homozygous transgeneticDrosophila lines with locustCYP408B1 andCYP409A1 were successfully constructed by using transgenic technology. Male transgenic flies (UAS-CYP408B1 and UAS-CYP409A1), and parental flies attp40 were crossed with tub-gal4 virgin flies, respectively. Genomic DNA and total RNA were isolated from hybrid offsprings: transgenic flies expressed locustCYP408B1 andCYP409A1 (tub>CYP408B1and tub>CYP409A1) and the control tub>attp40, respectively. At the same time, total RNA was used to synthesize cDNA using MLV reverse transcriptase. In order to validate transgenic flies UAS-CYP408B1 and UAS-CYP409A1, PCR amplification was conducted with DNA and cDNA as templates, respectively. The susceptibility of transgenicflies (tub>CYP408B1 and tub>CYP409A1) and control flies (tub>attp40, UAS-CYP408B1, UAS-CYP409A1 and attp40) to three selected insecticides (deltamethrin, malathion and chlorpyrifos) were analyzed. Cytochrome P450 monooxygenase activity of transgenic flies (tub>CYP408B1 and tub>CYP409A1) and the control tub>attp40 by fluorescence measurements were measured.[Result]PCR amplification showed a clear single target band in flies tub>CYP408B1 (1 555 bp) and tub>CYP409A1 (1 585 bp), respectively. RT-PCR and RT-qPCR showed the locustCYP408B1 andCYP409A1 were expressed in transgenicDrosophilaflies, respectively. Insecticide bioassay results indicated that the sensitivity of transgenic flies tub>CYP408B1 and tub>CYP409A1 to deltamethrin was higher than the control groups and that the resistance ratios were 1.59 and 1.83 compared with tub>attp40, respectively. However, the sensitivity of flies to malathion and chlorpyrifos did not increase. Measurements of cytochrome P450 monooxygenase activity showed that transgenic flies (tub>CYP408B1 and tub>CYP409A1) had no significant differences compared with the control tub>attp40 by fluorescence quantitative determination.[Conclusion]The transgenicDrosophila lines UAS-CYP408B1 and UAS-CYP409A1 were successfully constructed by using transgenic technology.CYP408B1 andCYP409A1 play a role in the deltamethrin detoxification.%【目的】细胞色素P450（Cytochrome P450）是昆虫体内广泛分布的代谢解毒酶系，飞蝗（Locusta migratoria）是重要农业害虫，揭示其体内P450代谢解毒功能对飞蝗有效治理尤为必要。研究旨在将飞蝗2个P450基因CYP408B1和CYP409A1分别转入果蝇（Drosophila melanogaster）体中，建立转基因果蝇品系，测定果蝇对杀虫剂的敏感性，为进一步探索飞蝗CYP408B1和CYP409A1对杀虫剂的代谢解毒功能提供依据。【方法】采用转基因技术成功构建转飞蝗P450基因CYP408B1和CYP409A1的纯合果蝇品系；分别选择雄性转基因果蝇UAS-CYP408B1、UAS-CYP409A1和亲本果蝇attp40与tub-gal4的处女果蝇杂交，提取子一代启动目的基因CYP408B1和CYP409A1表达的转基因果蝇品系tub＞CYP408B1和tub＞CYP409A1以及对照组果蝇品系tub＞attp40的DNA和总RNA，并将RNA体外反转录为cDNA。分别以DNA和cDNA为模板，PCR方法验证转基因果蝇品系 UAS-CYP408B1和 UAS-CYP409A1；通过杀虫剂生物学测定，进一步分析转基因果蝇品系 tub＞CYP408B1、tub＞CYP409A1以及对照组tub＞attp40、UAS-CYP408B1、UAS-CYP409A1和attp40对溴氰菊酯、马拉硫磷和毒死蜱3种杀虫剂的敏感性；采用荧光法定量测定转基因果蝇品系tub＞CYP408B1、tub＞CYP409A1以及对照组tub＞attp40中细胞色素 P450的总酶活性。【结果】PCR扩增结果显示以DNA为模板，启动目的基因表达的转基因果蝇品系tub＞CYP408B1和tub＞CYP409A1中均可以检测到目的条带，大小分别为1555和1585 bp；以cDNA为模板进行RT-PCR和RT-qPCR，结果显示在转基因果蝇品系中飞蝗CYP408B1和CYP409A1均得到表达。对3种杀虫剂的生物测定结果显示转基因果蝇品系tub＞CYP408B1和tub＞CYP409A1对溴氰菊酯的抗药性均较对照组高，且与 tub＞attp40相比，抗性比分别为1.59和1.83，而对马拉硫磷和毒死蜱的抗性比并未提高。细胞色素P450总酶活性结果显示转基因果蝇品系tub＞CYP408B1和tub＞CYP409A1总酶活性与对照组 tub＞attp40相比，均无显著性差异。【结论】利用转基因技术成功构建了转飞蝗 P450基因CYP408B1和CYP409A1的果蝇品系UAS-CYP408B1和UAS-CYP409A1；飞蝗CYP408B1和CYP409A1均对溴氰菊酯具有一定的解毒功能。