关键词: 策略, 策略

Abstract: 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.