昆虫学报 ›› 2020, Vol. 63 ›› Issue (7): 843-850.doi: 10.16380/j.kcxb.2020.07.008

• 研究论文 • 上一篇    下一篇

转飞蝗羧酸酯酶基因果蝇品系的构建及其在有机磷农药代谢解毒中的作用

尹菲1, 杨洋2, 张徐波2, 张建珍2, 张建琴1,*
  

  1. (1. 山西大学中医药现代研究中心, 太原 030006; 2. 山西大学应用生物学研究所, 太原030006)
  • 出版日期:2020-07-20 发布日期:2020-07-29

Construction of transgenic Drosophila strains with carboxylesterase genes of Locusta migratoria (Orthoptera: Acrididae) and their roles in the metabolic detoxification of organophosphorus pesticides

YIN Fei1, YANG Yang2, ZHANG Xu-Bo2, ZHANG Jian-Zhen2, ZHANG Jian-Qin1,*   

  1.  (1. Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China; 2. Institute of Applied Biology, Shanxi University, Taiyuan 030006, China)
  • Online:2020-07-20 Published:2020-07-29

摘要: 【目的】在活体水平验证飞蝗Locusta migratoria羧酸酯酶基因LmCesA1和LmCesA2是否参与有机磷杀虫剂的代谢解毒。【方法】采用Gal4/UAS系统,借助转基因技术,构建两个转基因黑腹果蝇Drosophila melanogaster品系,选取3品系Gal4(act-Gal4, tub-Gal4和c601-Gal4)果蝇作为母本分别与两种转基因果蝇(UAS-LmCesA1和UAS-LmCesA2)以及一种亲本对照果蝇(RB0006{y v; attP40, y+})进行杂交。对子一代转基因果蝇从DNA和RNA水平进行验证,筛选出成功构建的品系。采用生物测定方法检测转基因果蝇与Gal4果蝇杂交后代对马拉硫磷的抗性。【结果】转基因果蝇DNA水平鉴定结果显示,转基因果蝇tub>LmCesA1和tub>LmCesA2中分别扩增到目的基因LmCesA1和LmCesA2,而对照组果蝇tub>attP40中未扩增到目的基因。转基因果蝇RNA水平的检测结果显示,这两个基因在相应的杂交后代中均有表达,表明转基因果蝇构建成功。目的基因在转基因果蝇成虫不同组织中的表达结果表明,两个目的基因LmCesA1和LmCesA2分别在转基因果蝇c601>LmCesA1和c601>LmCesA2的肠道中高表达; LmCesA1在c601>LmCesA1果蝇肠道中的表达量分别是脑和表皮中的7.6和16.7倍, LmCesA2在c601>LmCesA2果蝇肠道中的表达量分别是脑和表皮中的5.4和10.9倍。杀虫剂生物测定结果显示,与对照组果蝇(c601>attP40)相比,超表达LmCesA2的果蝇(c601>LmCesA2)对马拉硫磷的抗性显著提高,抗性倍数为1.67。【结论】本研究的结论与我们前期采用RNAi结合杀虫剂生测的研究结论一致,即羧酸酯酶基因LmCesA2可能参与飞蝗对马拉硫磷的代谢解毒过程。

关键词: 飞蝗, 黑腹果蝇, 羧酸酯酶基因, 转基因, 杀虫剂, 代谢解毒, 抗药性

Abstract:  【Aim】 To determine whether the carboxylesterase genes LmCesA1 and LmCesA2 of Locusta migratoria are involved in the metabolic detoxification of organophosphorus insecticides in vivo. 【Methods】 Two transgenic strains of Drosophila melanogaster were constructed by using Gal4/UAS system and transgenic technology. Three Gal4 strains (act-Gal4, tub-Gal4 and c601-Gal4) were selected to hybridize with transgenic Drosophila strains (UAS-LmCesA1 and UAS-LmCesA2) and the reference strain (RB0006{y v; attP40, y+}). The offspring of transgenic strains was verified at the DNA and RNA levels, and then the successfully constructed strains were screened out. The resistance of the offspring of the transgenic strains and Gal4 strains to malathion was detected by bioassay. 【Results】 The identification results of transgenic Drosophila at the DNA level showed that the target genes LmCesA1 and LmCesA2 were amplified in the transgenic strains tub>LmCesA1 and tub>LmCesA2, respectively, but not in the reference group (tub>attP40), while those at the RNA level revealed that the two genes were expressed in the corresponding hybrid offspring, indicating that the transgenic Drosophila strains were successfully constructed. Tissue expression profiles of the two target genes in adults of the transgenic Drosophila showed that LmCesA1 and LmCesA2 were highly expressed in the gut of the transgenic strains c601>LmCesA1 and c601>LmCesA2, respectively. In the transgenic strain c601>LmCesA1 the expression level of LmCesA1 in the gut was 7.6- and 16.7-fold as high as those in the brain and cuticle, respectively, while in the transgenic strain c601>LmCesA2 the expression level of LmCesA2 in the gut was 5.4- and 10.9-fold as high as those in the brain and cuticle, respectively. The results of insecticide bioassay demonstrated that compared with the F1 generation of hybrid strain in the reference group (c601>attP40), the hybrid offspring overexpressing LmCesA2 (c601>LmCesA2) showed significantly enhanced resistance to malathion, with the resistance ratio of 1.67. 【Conclusion】 The conclusion of this study is consistent with that of our previous research by RNAi combined with insecticide bioassay, i.e., LmCesA2 may be involved in the metabolic detoxification of malathion in L. migratoria.

Key words: Locusta migratoriaDrosophila melanogaster, carboxylesterase gene, transgene, insecticide, metabolic detoxification, pesticide resistance