| 分子生物学手段检测转Bt基因棉花中Cry杀虫蛋白的定性和定量方法 |
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| 引用本文： | 李衍亮,方志翔,游静.分子生物学手段检测转Bt基因棉花中Cry杀虫蛋白的定性和定量方法[J].分析测试技术与仪器,2014(3):148-154. |
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| 作者姓名： | 李衍亮 方志翔 游静 |
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| 作者单位： | 中国科学院 广州地球化学研究所, 有机地球化学国家重点实验室, 广州 510640;广东省农业科学院 农业资源与环境研究所, 农业部南方植物营养与肥料重点实验室/广东省养分资源循环利用与耕地保育重点实验室, 广州 510640;中国科学院大学, 北京 100049;环境保护部 南京环境科学研究所, 南京 210042;中国科学院 广州地球化学研究所, 有机地球化学国家重点实验室, 广州 510640 |
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| 基金项目： | 本文系国家转基因专项（2013ZX08011-002，2014ZX08011-002）资助。 |
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| 摘 要： | 建立了转Bt基因棉花中Cry杀虫蛋白的提取、样品前处理以及酶联免疫(ELISA)定量分析方法,并使用凝胶电泳、普通聚合酶链式反应(PCR)和实时荧光定量PCR等分子生物学手段对转基因棉花中的Bt基因进行定性和定量检测.所建立的苏云金芽孢杆菌杀虫晶体蛋白(Cry1Ab蛋白和Cry1Ac蛋白)标准曲线线性关系良好,相关系数r2均大于0.999,相对标准偏差RSD均小于2.0%.方法简单、快速、重现性和精密度好,可为农业食品行业和环境领域科研人员提供一种简便快速地从转基因棉花中检测Bt毒蛋白的分析方法.
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| 关 键 词： | 转Bt基因棉花 Cry杀虫蛋白 酶联免疫法 凝胶电泳 实时荧光定量PCR |
| 收稿时间： | 2014/8/28 0:00:00 |
| 修稿时间： | 2014/9/12 0:00:00 |
Molecular Biological Methods for Qualitative and Quantitative Analysis of Cry Insecticidal Proteins in Transgenic Bt Cotton |
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| LI Ya-liang,FANG Zhi-xiang and YOU Jing.Molecular Biological Methods for Qualitative and Quantitative Analysis of Cry Insecticidal Proteins in Transgenic Bt Cotton[J].Analysis and Testing Technology and Instruments,2014(3):148-154. |
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| Authors: | LI Ya-liang FANG Zhi-xiang and YOU Jing |
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| Institution: | State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China;Key Laboratory of Plant Nutrition and Fertilizer in South Region of Ministry of Agriculture/Guangdong Key Laboratory of;Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection of China, Nanjing 210042, China;State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China |
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| Abstract: | An enzyme linked immunosorbent assay(ELISA) quantitative method has been established to analyze Cry proteins extracted from transgenic Bt cotton. Various molecular biological methodologies such as gel electrophoresis, ordinary polymerase chain reaction(PCR) and real-time fluorescence quantitative PCR were applied to detect Bt gene in transgenic cotton qualitatively and quantitatively. Both standard curves of Bacillus thuringiensis insecticidal crystal protein Cry1Ab and Cry1Ac proteins in the ELISA analysis were of good linearity with correlation coefficients(r2) of > 0.999, and the relative standard deviations(RSD) for the analyses were less than 2%. The newly established method is easy to operate, rapid, reproducible and precise, and it can be used in the fields of agro-food industry and environmental science to analyze Bt toxin proteins in genetically modified crops. |
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| Keywords: | transgenic Bt cotton Cry insecticidal protein ELISA gel electrophoresis real-time fluorescence quantitative PCR |
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