转耐盐基因水稻的PCR检测方法及其分子辅助育种

PCR Detection System for Genetically Modified Rice and Its Application in Genetic Breeding

研究采用改良CTAB法和磁珠自动提取法提取水稻种子基因组DNA,通过对水稻内源SPS基因、ThST3和Ubiquit-in启动子间构建特异序列进行PCR扩增,扩增产物结合排枪凝胶电泳实现快速检测。其中PCR扩增内源SPS基因的结果表明,采用改良CTAB法和磁珠自动提取法可用于市售水稻种子和转基因种子的DNA提取。实验合成的构建特异引物以及建立的PCR扩增反应体系能特异性地检测转耐盐基因水稻Theli。该方法检测灵敏度高,绝对检测低限达17.3×10-2ng,相对检测低限为0.41%,能有效地对转基因水稻ThST 3进行鉴定;稳定性好,可完全满足转基因水稻的定性检测、监督和标识管理需要。同时可用于对转基因水稻的辅助选择(MAS)育种。

A developed CTAB method and magnetic beads automatic extraction method were used to extract DNA from rice seed. Specific primers for PCR detection of genetically modified rice were designed to amplify endogenous SPS gene sequence and constructing specific segments, ThST 3 gene and Ubiquitin promoter, and corresponding detection methods were developed. Results of PCR amplification of SPS gene indicated that both the developed CTAB method and magnetic beads automatic extraction method were useful for DNA extraction from genetically modified rice seed and the sold rice seed. Results of amplification of the constructing specific segments （TheST 3-Ubi） indicated that the developed method was useful to detect the transgenic rice Theli seed, and the absolute and relative limit of detection （LOD） of the method got to 17.3 × 10 -2ng and 0.41% respectively. The developed PCR method can detect transgenic rice Theli seed specially and detective sensitiv- ity. And the method can satisfy the need of qualitative detection for genetically modified rice. Establishment of PCR Detection System can be used for marker-assisted selection （MAS） breeding.