棉铃虫ABC转运蛋白ABCG1的基因克隆、亚细胞定位及其与Cry1Ac毒力的关系

doi:10.16380/j.kcxb.2017.03.007

昆虫学报 ›› 2017, Vol. 60 ›› Issue (3): 297-308.doi: 10.16380/j.kcxb.2017.03.007

棉铃虫ABC转运蛋白ABCG1的基因克隆、亚细胞定位及其与Cry1Ac毒力的关系

公玲玲^{1，2，3，4}, 张丹丹², 郑曰英³, 于佃平³, 肖玉涛^2，3，4, 曲爱军^1，*

（1. 山东农业大学植物保护学院, 山东泰安 271018; 2. 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京 100193; 3. 夏津县农业局, 山东德州 253200; 4. 中国农业科学院深圳农业基因组研究所, 广东深圳 518120）

出版日期:2017-03-20 发布日期:2017-03-20

Gene cloning and subcellular localization of ATP-binding cassette transporter ABCG1 from Helicoverpa armigera (Lepidoptera: Noctuidae) and its relationship with Cry1Ac toxicity

GONG Ling-Ling^{1，2，3，4}, ZHANG Dan-Dan², ZHENG Yue-Ying³, YU Dian-Ping³, XIAO Yu-Tao^2，3，4, QU Ai-Jun^1，*

(1. College of Plant Protection, Shandong Agricultural University, Tai’an, Shandong 271018, China; 2. State Key Laboratory of Insect Pests and Plant Diseases, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 3. Agricultural Bureau of Xiajin County, Dezhou, Shandong 253200, China; 4. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, Guangdong 518120, China)

Online:2017-03-20 Published:2017-03-20

摘要/Abstract

摘要： 【目的】长期种植转Bacillus thuringiensis (Bt）基因作物使一些靶标害虫产生了Bt抗性，有研究表明小菜蛾Plutella xylostella 的white基因表达下调使其产生了Cry1Ac抗性，由于Pxwhite蛋白与ABC转运蛋白ABCG1同属于ABCG亚家族，我们推测棉铃虫Helicoverpa armigera ABCG1基因(HaABCG1)的表达下调可能与棉铃虫对Cry1Ac毒素的抗性有关。【方法】克隆并分析HaABCG1的开放阅读框(open reading frame， ORF)，通过构建HaABCG1基因的表达载体检测HaABCG1蛋白在TnH5细胞中的亚细胞定位；通过细胞毒力实验验证HaABCG1蛋白与Cry1Ac毒素的关系；利用RNAi技术验证HaABCG1表达下调是否会降低棉铃虫对Cry1Ac毒素的敏感性。【结果】棉铃虫ABCG1基因的开放阅读框长1 896 bp，编码蛋白含631个氨基酸残基，分子质量估计为69.63 kD。离体昆虫细胞表达的HaABCG1蛋白主要定位在细胞的核膜和内质网。通过RNA干扰和生物测定实验发现HaABCG1下调表达不能使棉铃虫在Cry1Ac毒素浓度为0.05 μg/mL的人工饲料上正常生长，3 d后处理组和对照组棉铃虫幼虫的体重变化无显著差异。经过细胞毒力实验证明HaABCG1蛋白不介导Cry1Ac毒素对TnH5细胞的毒力，它既不是Cry1Ac毒素的受体，也不是其他3种Bt毒素Cry1Ca， Cry2Aa和Cry1Fa的受体。【结论】HaABCG1基因表达下调与棉铃虫对Cry1Ac的抗性不相关，HaABCG1不是Cry1Ac毒素的受体。这是首次报道ABCG1基因不参与棉铃虫的Cry1Ac抗性。

关键词: 棉铃虫, ABCG1, Cry1Ac, Bt毒素受体, 亚细胞定位, Bt抗性

Abstract: 【Aim】 Cry resistance developed in target pests has been caused by the sustainable planting of Bt crops. Previous work indicated that the down-regulation of white gene is tightly linked to Cry1Ac resistance in Plutella xylostella, and Pxwhite and ATP-binding cassette transporter ABCG1 belong to ABCG subfamily, so we predict that the similar change of ABCG1 gene expression may be associated with the Cry1Ac resistance in Helicoverpa armigera. 【Methods】 The open reading frame (ORF) of HaABCG1 was cloned from H. armigera, the subcellular localization of HaABCG1 protein in TnH5 cells was detected by constructing expression vector of HaABCG1, the ABCG1 protein as the receptor of Cry1Ac toxin with cytotoxicity of Bt toxin was confirmed by cytotoxicity tests, and whether the down-regulation of HaABCG1 could decrease the susceptibility of H. armigera to Cry1Ac toxin was confirmed by RNA interference (RNAi) and bioassay. 【Results】 The open reading frame of HaABCG1 is 1 896 bp, encoding 631 amino acid residues with the predicted molecular weight of 69.63 kD. HaABCG1GFP is mainly localized on the nuclear membrane and endoplasmic reticulum of TnH5 cells. RNAi-mediated suppression of HaABCG1 expression did not decrease the susceptibility of H. armigera to Cry1Ac toxin, and the body weight change had no significant difference between the experimental group and the control group reared on artificial diet containing 0.05 μg/mL Cry1Ac for 3 d after RNAi. HaABCG1 protein did not mediate the cytotoxicity of Cry1Ac to TnH5 cells, and it was neither the receptor of Cry1Ac, nor the receptor of Cry1Ca, Cry2Aa or Cry1Fa. 【Conclusion】 The results suggest that the down-regulation of HaABCG1 is not associated with Cry1Ac resistance in H. armigera, and HaABCG1 is not the receptor of Cry1Ac. This is the first report that ABCG1 gene is not involved in the Cry1Ac resistance of H. armigera.

Key words: Helicoverpa armigera, ABCG1, Cry1Ac, Bt toxin receptor, subcellular localization, Bt resistance

公玲玲, 张丹丹, 郑曰英, 于佃平, 肖玉涛, 曲爱军. 棉铃虫ABC转运蛋白ABCG1的基因克隆、亚细胞定位及其与Cry1Ac毒力的关系[J]. 昆虫学报, 2017, 60(3): 297-308.

GONG Ling-Ling, Zhang Dan-Dan, Zheng Yue-Ying, Yu Dian-Ping, Xiao Yu-Tao, Qu Ai-Jun. Gene cloning and subcellular localization of ATP-binding cassette transporter ABCG1 from Helicoverpa armigera (Lepidoptera: Noctuidae) and its relationship with Cry1Ac toxicity[J]. Acta Entomologica Sinica, 2017, 60(3): 297-308.

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http://www.insect.org.cn/CN/Y2017/V60/I3/297

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棉铃虫ABC转运蛋白ABCG1的基因克隆、亚细胞定位及其与Cry1Ac毒力的关系

Gene cloning and subcellular localization of ATP-binding cassette transporter ABCG1 from Helicoverpa armigera (Lepidoptera: Noctuidae) and its relationship with Cry1Ac toxicity

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