[Objective] This study aimed to establish an effective detection technology for rapidly identifying Bt transgenic sugarcane, promptly removing non-Bt transgenic plants and increasing breeding efficiency of Bt transgen...[Objective] This study aimed to establish an effective detection technology for rapidly identifying Bt transgenic sugarcane, promptly removing non-Bt transgenic plants and increasing breeding efficiency of Bt transgenic plants, thereby improving breeding efficiency of insect-resistant sugarcane cultivars in China. [ Method] Approximately 1 mg of root tips and other materials were crushed, placed in the bottom of PCR tubes, added successively with solution I and mineral oil, headed at 95 ℃ for 15 rain, added with solution Ⅱ, and finally added with solution III containing GBt primer for PCR. After separated by using electrophoresis on agarese gel, amplification products were observed and photographed under a gel imaging system. [ Result] Various materials treated with different concentrations of solution led to different amplification results. Specifically, the appropriate concentration of NaOH in solution I was 0.1 -0.2 mol/L; the appropriate pH value of solution II was 2. No bands were amplified from mature leaves, tender leaves and old roots of Bt transgenic sugarcane, while root tips successfully amplified 545 bp target bands. The established method was used to identify and analyze 63 hybrid seedlings of YT 91 -976 × SBR216, results showed that a total of 17 samples exhibited 545 bp bands, accounting for approximately 27% of the total number of hybrid seedlings, indicating that these 17 seedlings were Bt transgenic sugarcane plants. [ Conclusion] The rapid detection method established in this study is conducive to identifying Bt transgenic sugarcane and improving breeding efficiency of insect-resistant sugarcane lines, which provides basis for screening of other transgenic materials.展开更多
转基因检测技术的标准化可以为转基因法规的制定和转基因产品的标识提供技术支持。采用碱裂解法快速提取鼠尾基因组DNA,用PCR方法检测其中的外源基因结构如cytomegalovirus(CMV)启动子、hGH polyA终止子及目的基因跨膜蛋白66(Transmembr...转基因检测技术的标准化可以为转基因法规的制定和转基因产品的标识提供技术支持。采用碱裂解法快速提取鼠尾基因组DNA,用PCR方法检测其中的外源基因结构如cytomegalovirus(CMV)启动子、hGH polyA终止子及目的基因跨膜蛋白66(Transmembrane protein 66,Tmem66),筛选到阳性样品。优化了多重PCR引物退火温度及缓冲液浓度,并探讨了扩增速率对多重PCR的影响。结果表明,此多重PCR方法可得到很好的扩增效果,可用于转基因小鼠外源基因的检测,同时为哺乳动物检测标准的建立提供参考。展开更多
基金Supported by Modern Agricultural Industry Technology System (CARS-2004B)
文摘[Objective] This study aimed to establish an effective detection technology for rapidly identifying Bt transgenic sugarcane, promptly removing non-Bt transgenic plants and increasing breeding efficiency of Bt transgenic plants, thereby improving breeding efficiency of insect-resistant sugarcane cultivars in China. [ Method] Approximately 1 mg of root tips and other materials were crushed, placed in the bottom of PCR tubes, added successively with solution I and mineral oil, headed at 95 ℃ for 15 rain, added with solution Ⅱ, and finally added with solution III containing GBt primer for PCR. After separated by using electrophoresis on agarese gel, amplification products were observed and photographed under a gel imaging system. [ Result] Various materials treated with different concentrations of solution led to different amplification results. Specifically, the appropriate concentration of NaOH in solution I was 0.1 -0.2 mol/L; the appropriate pH value of solution II was 2. No bands were amplified from mature leaves, tender leaves and old roots of Bt transgenic sugarcane, while root tips successfully amplified 545 bp target bands. The established method was used to identify and analyze 63 hybrid seedlings of YT 91 -976 × SBR216, results showed that a total of 17 samples exhibited 545 bp bands, accounting for approximately 27% of the total number of hybrid seedlings, indicating that these 17 seedlings were Bt transgenic sugarcane plants. [ Conclusion] The rapid detection method established in this study is conducive to identifying Bt transgenic sugarcane and improving breeding efficiency of insect-resistant sugarcane lines, which provides basis for screening of other transgenic materials.
文摘转基因检测技术的标准化可以为转基因法规的制定和转基因产品的标识提供技术支持。采用碱裂解法快速提取鼠尾基因组DNA,用PCR方法检测其中的外源基因结构如cytomegalovirus(CMV)启动子、hGH polyA终止子及目的基因跨膜蛋白66(Transmembrane protein 66,Tmem66),筛选到阳性样品。优化了多重PCR引物退火温度及缓冲液浓度,并探讨了扩增速率对多重PCR的影响。结果表明,此多重PCR方法可得到很好的扩增效果,可用于转基因小鼠外源基因的检测,同时为哺乳动物检测标准的建立提供参考。
