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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。
The genomic DNA of rice seeds was extracted by modified CTAB method and magnetic beads extraction method. The PCR products were amplified by specific PCR amplification of endogenous SPS gene, ThST3 and Ubiquitin promoter, and the amplified products were combined with discharge gel electrophoresis Quick check. PCR amplification of endogenous SPS gene results showed that the modified CTAB method and automatic magnetic beads can be used for the extraction of commercially available rice seeds and transgenic seeds of DNA. The constructed synthetic specific primers and the established PCR amplification reaction system can specifically detect the salt-tolerant transgenic rice Theli. The method has the advantages of high detection sensitivity, an absolute detection limit of 17.3 × 10-2ng and a relative detection limit of 0.41%, which can effectively identify the transgenic rice ThST 3 and has good stability and can fully meet the requirements of qualitative detection and supervision of the transgenic rice And logo management needs. At the same time, it can be used for MAS assisted breeding of transgenic rice.