Construction and Molecular Evaluation of A Positive Plasmid pUC18-RICE-screen for Screening Detection of Transgenic Rice (Oryza sativa)
MA Hui1, JIAO Xiao-Yu1, XU Xue1, LI Xia-Ying2, CHEN Zi-Yan2, WU Shuang1, PAN Wei-Qin1, ZHANG Xiu-Jie2,*, WANG Xiu-Feng1,*
1 Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China; 2 Development Center for Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100176, China
Abstract：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 materials of genetically modified rice, and also provides technical support for solving the problem of lack of positive standards for GM rice.
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