Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

作     者：ZHANG Xiujie JIN Wujun XU Wentao LI Xiaying SHANG Ying LI Sha OUYANG Hongsheng 

作者机构：College of Animal Sciences Jilin University Changchun 130062 China Development Center of Science and Technology Ministry of Agriculture and Rural Affairs Beijing 100176 China Biotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing 100081China College of Food Science and Nutritional EngineeringChina Agricultural University Beijing 100083China 

出 版 物：《Rice science》 (水稻科学（英文版）)

年 卷 期：2019年第26卷第4期

页      面：248-256,I0006,I0007页

核心收录：

学科分类：09[农学] 

主　　题：endogenous reference gene rice genetically modified crop phosphoenolpyruvate carboxylase gene sucrose-phosphate synthase gene phospholipase D gene starch branching enzyme 4 gene rice root-specific GOS9 gene 

摘      要：Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase (SPS), phospholipase D (PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs, a new ERG gene, phosphoenolpyruvate carboxylase (PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of grea