摘要
苏云金芽胞杆菌基因是转基因抗虫作物中通用的外源功能基因,在绝大多数抗虫转基因作物中均有存在,然而Bt基因检测标准样品的缺乏却限制了我国转Bt基因抗虫作物检测工作的发展。为了弥补传统基体标准样品的缺失,首先将Cry1Ab、Cry1Ac、Cry3A 3种常用Bt外源基因克隆到pUC57质粒上,通过测序、酶切和qPCR等技术对质粒的序列和扩增功能进行了验证,然后对扩增效率和实际应用情况加以测试,评价其转基因检测的适用性,构建了质粒标准分子。结果显示,制备的质粒标准分子测序结果与靶标序列完全符合,酶切结果、qPCR扩增结果和扩增效率等均符合预期,在Cry1Ab、Cry1Ac、Cry3A基因特异性检测中的应用符合阳性对照要求,表明制备的阳性质粒标准分子能够作为转Cry1Ab、Cry1Ac、Cry3A基因qPCR基因特异性检测的阳性标准样品。
The Bacillus thuringiensis gene is a universal foreign functional gene in transgenic insect-resistant crops.It exists in most insect-resistant transgenic crops.However,the lack of Bt gene detection standard materials limits the development of the detection on crops with transgenic insect-resistant Bt genes in China.In order to make up for the lack of traditional matrix standard materials,this experiment first cloned the three commonly used Bt foreign genes of Cry1Ab,Cry1Ac and Cry3A into the pUC57 plasmid,and carried out the sequence and amplification function of the plasmid by means of sequencing,enzyme digestion and qPCR.Validation,and then test the amplification efficiency and actual application conditions to evaluate the ap-plicability of the genetically modified detection.The results showed that the sequencing results of the plasmid prepared in this study are in complete agreement with the target sequence,and the results of enzyme digestion,qPCR amplification results and amplification efficiency are all in line with expectations.The application in the specific detection of Cry1Ab,Cry1Ac and Cry3A gene events is in line with the positive control.The requirements indicated that the positive plasmid reference molecules prepared in this study can be used as positive reference materials for the specific detection of transgenic Cry1Ab,Cry1Ac and Cry3A gene qPCR events.
作者
王颢潜
黄炎
石雨婷
朱鹏宇
谢宇宙
黄春蒙
卢小雨
付伟
WANG Haoqian;HUANG Yan;SHI Yuting;ZHU Pengyu;XIE Yuzhou;HUANG Chunmeng;LU Xiaoyu;FU Wei(Development Center for Science and Technology,Ministry of Agriculture and Rural Affairs,Beijing 100176,China;Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Beijing Health Center Acadamy,Beijing 101149,China;China Association for Standardization,Beijing 100048,China;Chinese Academy of Inspection and Quarantine Center for Biosafety,Hainan Sanya 572025,China;Animal and Plant Inspection and Quarantine Technology Centre,Guangdong Shenzhen 518038,China)
出处
《生物技术进展》
2023年第1期83-89,共7页
Current Biotechnology
基金
海南省重大科技计划项目(ZDKJ202002)
中国检科院基本科研业务费(2020JK016)。
