苦瓜基因组DNA的提取及ISSR扩增体系的优化

为了快速获取高质量的苦瓜基因组DNA，以便进行苦瓜白粉病抗性基因分子标记研究，比较了不同的DNA提取方法、不同部位的苦瓜叶片提取基因组DNA的产量和质量，探究了苦瓜基因组DNA提取的最佳方法和叶片的最适部位；研究了ISSR-PCR的退火温度，并采用正交设计法对影响ISSR-PCR的Mg²⁺、dNTPs、Taq酶、模板DNA以及引物浓度等5个因素进行了优化。结果表明，采用改良CTAB法从苦瓜顶端嫩叶中提取的基因组DNA OD260/OD280值在1.8~1.9之间，OD260/OD230值为2.0左右，对DNA样品进行琼脂糖凝胶电泳检测，主带清晰，降解较少，产量和纯度均较高，效果较好。优化后的ISSR-PCR反应体系为：2.5μL 10×PCR buffer，2.5 mmol/L MgCl₂，250μmol/L dNTPs，10 ng模板DNA，0.75 U Taq酶，引物浓度0.7μmol/L，反应总体积为25μL，引物UBC826最佳退火温度为53℃。该体系在多次重复中均能获得良好的扩增结果。苦瓜基因组DNA提取的最佳方法为改良CTAB法，最适合的部位为顶端嫩叶。

The Extraction of the Genomic DNA in Bitter Gourd and Optimization of ISSR Amplified System

In order to rapid acquiring high quality genomic DNA of Bitter gourd (Momordica charantia L.) and research the resistance gene molecular marker of powdery mildew (Sphaerotheca fuliginea), the paper compared the extracted genomic DNA’s yield and quality by different extracted methods and different physiological periods’leaves of bitter gourd to search the best method and the most suitable period in extracting genomic DNA from bitter gourd. The annealing temperatures of ISSR-PCR (inter-simple sequence repeat and polymerase chain reaction) was researched, and the concentrations of Mg²⁺ , dNTPs, Taq DNA polymerase, genomic DNA and primers which effect ISSR-PCR were optimized by using orthogonal design method. The result showed that the OD260/OD280 values of the genomic DNA extracted from the top leaves of bitter gourd by improved CTAB method were 1.8-2.0 and the OD260/OD230 values were about 2.0, the electrophoretogram showed that the primary brands were clear and less degradation, the DNA samples were pure and had good quality, the effect was the best. The optimal system was 2.5μL 10×PCR buffer, 2.5 mmol/L MgCl₂, 250μmol/L dNTPs, 10 ng genomic DNA, 0.75 U Taq DNA polymerase, 0.7μmol/L primers, and the total volume of the reaction was 25μL, the most compatible annealing temperatures of primer UBC826 was 53℃. The optimal system could be employed to gain good results in repeated PCR experiments. The best extracting method was the improved CTAB method and the top tender leaf was the most fitting.