Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (6): 1103-1111.doi: 10.3864/j.issn.0578-1752.2015.06.07

• PLANT PROTECTION • Previous Articles     Next Articles

Susceptibility of Transgenetic Drosophila with Locusta migratoria CYP408B1 and CYP409A1 to Insecticides

GAO Cui-e1,3, ZHANG Jian-qin2, ZHANG Xu-bo1,3, ZHANG Jian-zhen1,3, FENG Yun-tao3,4, WU Hai-hua1,3, MA En-bo1,3   

  1. 1Research Institute of Applied Biology, Shanxi University, Taiyuan 030006
    2Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006
    3Shanxi Key Laboratory of Integrated Pest Management in Agriculture, Taiyuan 030006
    4Institute of Plant Protection, Shanxi Academy of Agricultural Science, Taiyuan 030032
  • Received:2014-09-27 Online:2015-03-16 Published:2015-03-16

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Abstract: 【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 transgenic Drosophila melanogaster lines with locust CYP408B1 and CYP409A1 genes, determine the susceptibility of transgenic Drosophila expressed exogenous CYP408B1 or CYP409A1 to insecticides, and to reveal the characteristics of locust CYP408B1 and CYP409A1. 【Method】 Homozygous transgenetic Drosophila lines with locust CYP408B1 and CYP409A1 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 locust CYP408B1 and CYP409A1 (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 locust CYP408B1 and CYP409A1 were expressed in transgenic Drosophila flies, 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 transgenic Drosophila lines UAS-CYP408B1 and UAS-CYP409A1 were successfully constructed by using transgenic technology. CYP408B1 and CYP409A1 play a role in the deltamethrin detoxification.

Key words:  transgenic Drosophila, Locusta migratoria, CYP408B1 and CYP409A1, insecticide bioassay, enzyme activity

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