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Construction and Application of Standard Plasmid Molecules for Screening Detection of Four Major Genetically Modified Crops |
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Abstract Plasmid molecule worked as reference material has been shown to be a good alternative in genetically modified organisms (GMOs) detection, and can promptly satisfy the regulatory requirements of GMOs. In this study, according to the transgenic information of 4 main crops including rice (Oryza sativa L.), maize (Zea mays L.), soybean (Glycine max) and rape (Brassica napus), 4 types of plasmids for GMO screening were developed and confirmed. The constructed pMD-rice contained 7 target elements including promoter of Cauliflower mosaic virus (CaMV35S), terminator of nopaline synthase (tNOS) and selective marker genes, such as biolaphos resistance (Bar), neomycin phosphotransferase (NPTⅡ) and hygromycin B phosphotransferase (Hpt), insect resistant gene of Bacillus thuringiensis (Bt) and a rice endogenous reference gene of sucrose phosphate synthase (SPS) were selected. The constructed pMD-maize was consisted of 4 target sequences including tNOS, CaMV35S, Bar and zSSⅡb as endogenous reference genes. The constructed pMD-soybean contained 5 target elements including CaMV35S, tNOS and 2 exogenous genes cowpea trypsin inhibitor gene (CpTi) and enolpyruvylshikimate phosphate synthase (EPSPS), and the Lectin precursor protein gene as endogenous reference gene. The constructed pMD-rape contained 8 target elements including CaMV35S, Figwort mosaicvirus 35S (FMV35S), nopaline synthase promoter (P-nos) and tobacco tapetum specific promoter (PTa29), tNOS, terminator-35S (T-35S) and the 7th gene of TL-DNA (g7) and high mobile group protein gene (HMG) as endogenous reference genes. In theory, all elements of the 4 plasmids covered 90% of the approved transgenic events of 4 major crops as maize, rape, soybean and rice in the world, and they covered 100% of transgenic events that had been approved in China. Then the constructed plasmids and reference materials were comparatively analyzed and all the results demonstrated that the constructed plasmids were suitable for GMO screening detection.
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Received: 23 March 2015
Published: 09 July 2015
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Clive James. 2015. 2014年全球生物技术/转基因作物商业化发展态势.中国生物工程杂志, 35(1): 1-14.Corbisier P, Broeders S, Charels D, et al. 2007. Certification of plasmidic DNA containing MON 810 maize DNA fragments, ERM-AD413 [R]. EC certification report, EUR 22948, ISBN 978-92-79-07139-3.Babekova R, Funk T, Pecoraro S, Engel K.H, Busch U. 2009. Development of an event-specific real-time PCR detection method for the transgenic Bt rice line KMD1, Eur. Food Res. Technol. 228: 707–716.Bajaj S, Mohanty A. 2005. Recent advances in rice biotechnology—towards genetically superior transgenic rice, Plant Biotechnol. J. 3: 275–307.Huang C C, Pan T M. 2005. Event-specific real-time detection and quantification of genetically modified roundup ready soybean [J]. J Agric Food Chem, 53: 3833-3839.Koppel R, Zimmerli F, Breitenmoser A. 2010. Multiplex real-time PCR for the simultaneous detection and quantification of DNA from three transgenic rice species and construction and application of an artificial oligonucleotide as reference molecule, Eur. Food Res. Technol. 230: 731–736.Wang W, Zhu T, Lai F, Fu Q. 2011. Event-specific qualitative and quantitative detection of transgenic rice Kefeng-6 by characterization of the transgene flanking sequence, Eur. Food Res. Technol. 232: 297–305.Wu G, Wu Y, Nie S, Zhang L, Xiao L, Cao Y, Lu C. 2010. Real-time PCR method for detection of the transgenic rice event TT51-1, Food Chem. 119: 417–422.Xiaofu Wang, Xiaoyun Chen, Junfeng Xu et al. 2014. Multiplex event-specific qualitative polymerase chain reaction for detecting three transgenic rice lines and application of a standard plasmid as a quantitative reference molecule. Analytical Biochemistry 464: 1–8.Xu J F, Wang X F, Chen X Y et al. 2011. Qualitative detection and quantification of a Cry1A(b) transgene present in rice cv. Zhejing 22, Eur. Food Res. Technol. 233: 259–266.Yang L, Guo J, PAN A, et al. 2007. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule [J]. Journal of Agricultural and Food Chemistry, 55 ( 1) : 15-24.曹际娟,徐君怡,赵昕,等. 2012. SN/T1196-2012. 转基因成分检测 玉米检测方法 [S]. 中华人民共和国国家质量监督检验检疫总局.段武德. 2009. 转基因植物检测 [M].中国农业出版社, 2009: 12 - 32.胡尚杰、吴刚、武玉花,等. 2010. 转基因作物筛查用阳性质粒分子的构建及应用研究. 中国油料作物学报, 32(2): 173-179.卢长明,宋贵文,吴刚,等. 2012. 农业部1782号公告-2-1012. 转基因植物及其产品成分检测 标记基因NPTII、HPT和PMI定性PCR方法[S]. 中华人民共和国农业部.路兴波,宋贵文,李凡,等. 2012. 农业部1782号公告-6-1012. 转基因植物及其产品成分检测 bar或pat基因定性PCR方法 [S]. 中华人民共和国农业部.罗云波,黄昆仑,张大兵,等. 2003. NY/T675-2003. 转基因植物及其产品检测-大豆定性PCR 方法 [S]. 中华人民共和国农业部.潘良文,沈禹飞,陈家华,等. 2003. SN/T1197-2003. 油菜籽中转基因成分定性PCR 检测方法 [S]. 中华人民共和国国家质量监督检验检疫总局.王永,宋贵文,兰青阔,等. 2012. 农业部1782号公告-7-2012. 转基因植物及其产品成分检测 CpTI基因定性PCR方法 [S].中华人民共和国农业部.谢家建,沈平,彭于发,等. 2012. 农业部1782号公告-3-1012. 转基因植物及其产品成分检测 调控元件CaMV35S启动子、FMV35S启动子、NOS启动子、NOS终止子和CaMV35S终止子定性PCR方法 [S]. 中华人民共和国农业部. |
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