转基因水稻筛查检测阳性质粒pUC18-RICE-screen的构建及分子评价  

Construction and Molecular Evaluation of A Positive Plasmid pUC18-RICE-screen for Screening Detection of Transgenic Rice(Oryza sativa)

作　　者：马卉[1] 焦小雨 许学 李夏莹 陈子言 吴爽[1] 潘伟芹 张秀杰 汪秀峰[1] MA Hui;JIAO Xiao-Yu;XU Xue;LI Xia-Ying;CHEN Zi-Yan;WU Shuang;PAN Wei-Qin;ZHANG Xiu-Jie;WANG Xiu-Feng(Rice Research Institute,Anhui Academy of Agricultural Sciences,Hefei 230031,China;Development Center for Science and Technology,Ministry of Agriculture and Rural Affairs,Beijing 100176,China)

机构地区：[1]安徽省农业科学院水稻研究所,合肥230031 [2]农业农村部科技发展中心,北京100176

出　　处：《农业生物技术学报》2020年第7期1177-1192,共16页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31601289);农业农村部科技发展中心项目“主要非授权转基因作物及其产品检测技术研究”安徽省科技重大专项(201903a06020012)。

摘　　要：标准物质是转基因生物安全监管和检验检测工作中进行质量控制的关键。目前,国内用于转基因检测的阳性对照资源匮乏,主要为基体标准物质,难以满足转基因生物安全监管和检测工作的需要。质粒标准物质作为转基因检测标准物质新的发展方向,具有广泛的应用价值。本研究通过收集整理相关数据库信息,获得了国内外19种主要转基因水稻(Oryza sativa)品系的遗传信息,全面分析其外源基因表达框中调控元件/基因的组成情况及使用频率,并结合目前现有转基因检测技术标准,确定了以水稻内标基因磷脂酶D基因(phospholipase D gene,PLD)、蔗糖磷酸合酶基因(sucrose phosphate synthase gene,SPS)和靶标基因CaMV35S启动子(35S promoter from Cauliflower mosaicvirus,PCaMV35S)、Ubiquitin启动子(ubiquitin promoter from maize,PUbiquitin)、CaMV35S终止子、NOS终止子(terminator of nopaline synthase gene,TNOS)、抗草丁膦除草剂基因(bialaphos resistance gene,Bar)、潮霉素磷酸转移酶基因(hygromycin phosphotransferase gene,HPT)、苏云金芽胞杆菌基因(Bacillus thuringiensis gene,Bt)等9个调控元件/基因用于构建阳性质粒pUC18-RICE-screen。该质粒通过人工合成技术获得,经酶切、测序、PCR验证,以及对检测不同筛查元件/基因的适宜质粒浓度进行综合评价,结果表明,该质粒可用于本研究所涉19个转基因水稻的筛查检测,且其作为阳性对照用于各筛查元件/基因PCR和qRT-PCR筛查检测的适宜浓度范围为1×103~1×105 copies/μL。本研究为转基因水稻安全监管提供了一种不依赖于转基因水稻原材料的阳性对照,也为解决转基因水稻阳性标准品匮乏问题供了技术支持。Reference materials are the key to quality control in the detection and supervision of genetically modified organisms(GMOs).However,at present,the positive controls for GMOs detection in China are mainly matrix reference materials and it's very limited,which is difficult to meet the needs of GMOs safety supervision and detection.As a new development direction of transgenic detection reference materials,the plasmid reference materials have wide application value.In this study,genetic information of 19 major transgenic rice(Oryza sativa)at home and abroad were obtained through collecting and collating relevant databases,By analyzing the composition and use frequency of the genetic elements in their foreign gene expression frames,and combining current technology standards of transgenic detection,a total of 9 regulatory elements/genes,including 2 rice endogenous reference gene of sucrose phosphate synthase gene(SPS)and sucrose phosphate synthase gene(SPS)and 7 targets,the CaMV35S promoter(35S promoter from Cauliflower mosaicvirus,PCaMV35S),Ubiquitin promoter(ubiquitin promoter from maize,PUbiquitin),CaMV35S terminator,NOS terminator(terminator of nopaline synthase,TNOS),bialaphos resistance gene(Bar),hygromycin phosphotransferase gene(HPT)and Bacillus thuringiensis gene(Bt)were used to construct the standard plasmid pUC18-RICE-screen.This plasmid was obtained by synthetic technology,verified by enzyme digestion,sequencing,PCR amplification,and the plasmid concentration suitable for detecting different regulatory elements/genes was comprehensively evaluated.The experiment results showed that the constructed plasmid could be used for the screening detection of 19 transgenic rice involved in this research,and the suitable concentration range was 1×103~1×105 copies/μL when it was used as a positive control for each screening element/gene PCR and qRT-PCR screening detection.This study provides a positive control for the safety regulation of genetically modified rice,which does not depend on the positive-control raw mat

关 键 词：转基因水稻 阳性质粒分子 调控元件 筛查检测 

分 类 号：S234[农业科学—农业机械化工程]