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Abstract Endogenous reference genes(ERGs) refer to a class of genes characterized by species specificity, low copies and allelic conservation. The specific PCR amplification of ERGs has important meaning for quantitatively detecting the foreign gene of transgenic crops. In this paper, after looking for present related article, 5 related endogenous reference genes (the rice root-specific gene(GOS9), the phospholipase D gene (PLD), the sucrose phosphate synthase gene (SPS), the rice starch branching enzyme gene (RBE4) and the Ubiquitin 5 gene(UBQ5)) and 2 exogenous reference genes(Cry1Ab and Cry1Ac/Cry1Ab) were selected. Transgenic rice KMD2 and TT51-1 were selected as template. By comparing the products of PCR of 5 related endogenous reference genes with the products of PCR of exogenous genes, the endogenous reference gene which PCR products are most close to exogenous gene PCR products was picked out. On the basis, more research on the selected endogenous reference gene was analyzed. Limitations in Real-time PCR applications to relative quantification of number of DNA targets had led to new developments such as the digital PCR(d-PCR) which allows accurate measurement of DNA copies without the need for a reference calibrator, so the ratio of copy number of KMD2/RBE4 and TT51-1/RBE4 were analyzed by digital PCR, Real-time PCR analysis on the amplification efficiency of two transgenic rice, the stability of PCR amplification and detection sensitivity. The results showed that compared to other endogenous reference genes, the amplification products of RBE4 was the most close to the amplification products of two exogenous genes, the ratio of copy number of two transgenic rice were close to 1∶1, 115.9% and 105.3%, respectively. The repeatability and stability of PCR system of RBE4 were also satisfied with the endogenous genes for quantify matrix reference materials of transgenic rice. In the case of the RBE4 TaqMan assay for two transgenic rice KMD2 and TT51-1, the limit of detection(LOD) were between 5 and 11 copies of TT51-1 and between 3 and 12 copies of KMD2 respectively, and the limit of quantitation(LOQ) are between 11 and 22 copies of TT51-1 and between 12 and 24 copies of KMD2, respectively. Therefore, RBE4 is as a proper endogenous gene for preparation of matrix reference materials of transgenic crops and quantitative detection of transgenic crops.
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Received: 03 May 2012
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SHEN Xue-Jing, ZHANG Er-Qin, XU Qian-Ru, LIU Lin-Ke, WAN Bo, LIU Yun-Chao, SUN Ya-Ning, ZHAO Dong, NIU Xiang-Xiang, DENG Rui-Guang, ZHANG Gai-Ping. Expression of Rabies virus G Protein in Rice (Oryza sativa) and Identification of Its Genetic Stability[J]. 农业生物技术学报, 2019, 27(2): 204-211. |
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