›› 2017, Vol. 60 ›› Issue (9): 1006-1012.doi: 10.16380/j.kcxb.2017.09.004

• 研究论文 • 上一篇    下一篇


王芮1, 张浩淼1, 邓蕾1, 肖倩倩1, 王秋霞1, 刘广娜2, 韩召军1, 吴敏1,*   

  1. ( 1. 南京农业大学植物保护学院, 南京 210095; 2. 吉林农业科技学院, 吉林 132101)
  • 出版日期:2017-09-20 发布日期:2017-09-20

Carboxylesterase LsCarE1 mediates insecticide resistance in Laodelphax striatellus (Hemiptera: Delphacidae)  

WANG Rui1, ZHANG Hao-Miao1, DENG Lei1, XIAO Qian-Qian1, WANG Qiu-Xia1, LIU Guang-Na2, HAN Zhao-Jun1, WU Min1, *   

  1. (1. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; 2. Jilin Agricultural Science and Technology University, Jilin 132101, China)
  • Online:2017-09-20 Published:2017-09-20

摘要: 【目的】我们前期的研究发现,在灰飞虱Laodelphax striatellus抗毒死蜱品系、抗吡虫啉品系和抗溴氰菊酯品系中的总酯酶活力都明显升高。本研究旨在筛选导致总酯酶活力升高的酯酶基因并验证其在抗药性中的作用。【方法】通过转录组测序和RT-qPCR比较抗、感灰飞虱品系雌成虫中各个酯酶基因的表达量;采用显微注射技术建立超量表达灰飞虱羧酸酯酶基因的果蝇品系;并利用生物测定技术检测过量表达灰飞虱酯酶基因的果蝇对毒死蜱、吡虫啉和溴氰菊酯的敏感性。【结果】通过转录组数据和RT-qPCR筛选到羧酸酯酶基因LsCarE1(GenBank登录号: HM600723)在3个灰飞虱抗性品系中均过量表达。将LsCarE1基因按黑腹果蝇Drosophila melanogaster偏好的密码子优化后构建转基因载体质粒pUAST-attB-LsCarE1Optimized,成功建立纯合的转基因果蝇品系UAS-LsCarE1Optimized。利用GAL4启动品系da-gal4与UAS-LsCarE1Optimized品系杂交,获得超量表达LsCarE1的果蝇品系da>LsCarE1Optimized。定量PCR检测发现,目标基因LsCarE1在超量表达品系da>LsCarE1Optimized中的表达量是对照品系UAS-LsCarE1Optimized中表达量的257.3倍;而另一对照品系da>gal4中没有检测到LsCarE1的表达。此外,黑腹果蝇内源的酯酶同源基因表达量显著低于目标基因的表达量。生物测定结果显示,与对照组da>gal4果蝇品系相比,启动超量表达LsCarE1的转基因果蝇da>LsCarE1Optimized对毒死蜱和吡虫啉的耐药性分别提高了4.19和2.46倍,但对溴氰菊酯的耐药性无显著变化。【结论】LsCarE1的过量表达与灰飞虱对毒死蜱和吡虫啉的抗性相关。  

关键词: 灰飞虱, 代谢抗性, 羧酸酯酶, 转基因果蝇, 超量表达

Abstract: 【Aim】 In our previous studies, the activities of total esterases were found to be significantly increased in the chlorpyrifos, imidacloprid and deltamethrin resistant strains of the small brown planthopper, Laodelphax striatellus. The aim of this study is to identify the carboxylesterase gene causing this increase and to verify its roles in insecticide resistance. 【Methods】 The expression levels of carboxylesterase genes in female adults of the resistant and susceptible strains of L. striatellus were detected by transcriptome sequencing and RT-qPCR. Microinjection was used to establish a transgenic Drosophila melanogaster strain overexpressing the target gene. Topical application method was used to investigate the responses of transgenic D. melanogaster overexpressing the target gene to chlorpyrifos, imidacloprid and deltamethrin, respectively. 【Results】 The expression level of LsCarE1 (GenBank accession no.: HM600723) was upregulated in female adults of the chlorpyrifos, imidacloprid and deltamethrin resistant strains of L. striatellus. Based on the sequence of LsCarE1, codon optimization was performed, and the open reading frame of LsCarE1 was cloned into the transgenic vector pUAST-attB. The transgenic homozygous strain UAS-LsCarE1Optimized was successfully established and then genetically crossed with the GAL4 driver line to obtain a da>LsCarE1Optimized strain that exhibited the overexpression of LsCarE1. The expression level of LsCarE1 in the da>LsCarE1Optimized strain was 257.3 times as high as that in the reference strain UAS-LsCarE1Optimized. The expression of LsCarE1 was not detected in the reference strain da>gal4. Furthermore, the expression levels of the endogenous esterase homologous genes in D. melanogaster were significantly lower than that of LsCarE1. The bioassay results indicated that as compared with the control strain da>gal4, the overexpression strain da>LsCarE1Optimized  showed increased tolerance to chlorpyrifos and imidacloprid by 4.19- and 2.46-fold, respectively, but showed no significantly increased tolerance to deltamethrin. 【Conclusion】 The overexpression of LsCarE1 is related to the increased resistance to chlorpyrifos and imidacloprid in L. striatellus.  

Key words: Laodelphax striatellus; metabolic resistance, carboxylesterase, transgenic Drosophila, overexpression