基于多靶标质粒分子的转基因大豆快速筛查方案  

A Rapid Protocol for Screening Genetically Modified Soybeans Based on Positive Plasmid Molecule Containing Multiple Targets

作　　者：史宗勇 刘璇 许冬梅 李夏莹 陈子言 梁晋刚 王颢潜 张雨琪 温洪涛 张秀杰 高建华 SHI Zong-Yong;LIU Xuan;XU Dong-Mei;LI Xia-Ying;CHEN Zi-Yan;LIANG Jin-Gang;WANG Hao-Qian;ZHANG Yu-Qi;WEN Hong-Tao;ZHANG Xiu-Jie;GAO Jian-Hua(Department of Bioinformatics,College of Life Sciences,Shanxi Agricultural University,Taigu 030801,Shanxi,China;Crops Ecological Environment Security Inspection and Supervision Center(Taiyuan),Ministry of Agriculture and Rural Affairs,Taigu 030801,Shanxi,China;Development Center for Science and Technology,Ministry of Agriculture and Rural Affairs,Beijing 100025,China;Quality and Safety Institute of Agricultural Products,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,China;Department of Bioscience,College of Life Sciences,Shanxi Agricultural University,Taigu 030801,Shanxi,China)

机构地区：[1]山西农业大学生命科学学院生物信息系,山西太谷030801 [2]农业农村部农作物生态环境安全监督检验测试中心(太原),山西太谷030801 [3]农业农村部科技发展中心,北京100025 [4]黑龙江农业科学院农产品质量安全研究所,哈尔滨150086 [5]山西农业大学生命科学学院生物科学系,山西太谷030801

出　　处：《中国生物化学与分子生物学报》2021年第11期1540-1554,共15页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家科技重大专项(No.2018ZX08012001-009)资助。

摘　　要：随着我国批准进口和自主研制的转基因大豆转化体数量不断增加,亟需建立覆盖全面、检测靶标数量较少且符合国情的快速筛查方案。另外,为了控制检测工作量,还需构建对应的多靶标质粒(multi-targets plasmid,MTP)分子,作为大豆转基因成分检测的阳性物质。本文通过收集整理相关转化体的分子特征信息,获得了29种转基因大豆独立转化体的外源转化元件信息。全面分析其组成情况及使用频率,同时结合我国转基因检测相关标准方法,从而确定一套新的筛查方案。该方案包括8个检测靶标,CaMV 35S启动子(P-35S)、NOS终止子(T-nos)、耐除草剂基因pat、E9终止子(T-E9)、抗虫基因cry1Ac、AHAS启动子(P-AHAS)、pinⅡ终止子(T-pinⅡ)和DP305423转化体的特征序列,以及1个大豆内标准基因Lectin。通过这9个靶标序列的检测,可全面筛查上述29种转基因大豆独立转化体。本文将其称为转基因大豆筛查的"8+1"方案。将这9种靶标的检测序列串联并接入pUC18质粒,获得MTP分子pDDSC-1910。为了验证该分子作为阳性物质的适用性,以其为模板,对9种靶标进行定性PCR分析,结果均能获得预期的特异性扩增产物,且表现出较高的灵敏度。因此,本文建立了一个覆盖全面的转基因大豆筛查新方案,并成功构建了相应的MTP分子作为阳性物质,为转基因大豆的高效筛查和检测工作提供便利。Recently we witness the rising number of genetically modified(GM)soybean(Glycine max)events approved for importing from abroad and developed domestically,so it is urgent to establish a rapid screening protocol that can cover more events with less detection targets and fit the national condition.Additionally,in order to control the detection workload,it is also necessary to construct a multi-targets plasmid(MTP)molecule that can be used as the positive material.In this study,the information of the transgenic elements in 29 GM soybean events was collected and the combinations and frequencies of these elements were analyzed,to establish a novel screening protocol.It includes eight detecting targets,CaMV35 S promoter(P-35 S),NOS terminator(T-nos),herbicide tolerance gene pat,E9 terminator(T-E9),insecticidal gene cry1 Ac,AHAS promoter(P-AHAS),pinⅡterminator(T-pinⅡ),and the eventspecific sequence of the transgenic event DP305423,and an endogenous reference gene of soybean Lectin.After validation,the 29 GM soybean events described above can be screened by detection of the nine targets.This is referred to as the"8+1"protocol for GM soybean screening.Then these targeted sequences described in the protocol were simultaneously inserted into a cloning vector to construct the corresponding MTP pDDSC-1910.Finally,we tested whether it could be a positive plasmid.As expected,PCR analysis using pDDSC-1910 as a template showed that specific amplicons were observed with high sensitivity.Therefore,the"8+1"screening protocol for GM soybean was established,and the positive plasmid molecule pDDSC-1910 containing corresponding targets was successfully constructed.These results would facilitate the efficient screening and detection of transgenic soybeans.

关 键 词：转基因大豆 筛查 外源元件 阳性物质 质粒 

分 类 号：S565.1[农业科学—作物学]