转基因甘蔗植株Southern杂交体系的优化

The Optimization of Southern Blot for Transgenic Sugarcane Plants

对甘蔗Southern杂交体系的优化,旨为转基因甘蔗Southern杂交鉴定分析提供参考。以转基因甘蔗为材料,就探针不同标记方法的比较、甘蔗基因组DNA的提取、基因组DNA酶切量、酶切时间及杂交过程等方面,对地高辛标记的Southern杂交技术进行了优化研究。结果表明,改良的CTAB法提取的甘蔗DNA能满足后期实验的要求;PCR法标记探针的效率较随机引物法标记探针的效率高,更适合用于Southern杂交;40μg的DNA在400μL酶切体系中,酶切10 h可获得良好的酶切效果;杂交温度40℃,杂交18 h,可获得清晰的杂交条带。

Using sugarcane as materials, the DIG-labeled Southern blot was optimized through several key aspects ：the comparison of different-labeled probe methods, extraction of sugarcane genome DNA, the amount of enzyme digestion of genome DNA, enzyme digestion time and a serial of procedures in the process of the hybrid. The results showed that sugarcane DNA extracted by the improved CTAB method met the requirements of the latter experiments, the efficiency of PCR-labeled probe was higher than that of random primer-labeled probe, so PCRlabeled probe method was suitable for the Southern blot, 40 μg DNA samples in enzyme digestion system of 400 μL for 10 hours could achieved desirable digestion result;while hybrid temperature was 40℃ and hybridizing time was 18 h, a clear hybridization band could be observed. The optimization of sugarcane Southern blot provides a reference for the analysis of Southern blot of transgenic sugarcane.