珍稀濒危植物永瓣藤DNA提取与ISSR条件优化

DNA extraction and optimization of ISSR reaction system for rare and endangered plant Monimopetalum chinense

以硅胶干燥叶片为材料，研究永瓣藤DNA的提取方法，并对影响简单重复序列区间分子标记反应的各条件进行了优化。建立了永瓣藤ISSR的优化反应体系和程序，即在20μL反应体系中，含20ng模板DNA、2．375 mmol／L Mg^2＋、0．15mmol／L dNTPs、1．5 U Taq DNA聚合酶、225nmol／L随机引物；扩增程序为：94℃预变性5min；然后94℃15s、48-50℃ 45 s、72℃1 min，35个循环，最后72℃延伸10min，4℃保存。本研究为进行永瓣藤种群遗传多样性研究奠定了基础。

In the present study, DNA of Monirnopetalum chinense was extracted from the silica gel dried leaves. The optimized reaction system and programme of inter simple sequence repeats（ISSR）for M. chinense are as follows：the reactions were performed in a volume of 20 μL containing 20 ng of template DNA, 2. 375 mmol/L Mg^2＋ , 0. 15 mmol/L dNTPs, 1.5U Taq DNA polymerase,225 nmol/L primer; thermal cycling was programmed by 1 cycle for 5 min at 94 ℃ ,followed by 35 cycles for 15 s at 94 ℃ ,45 s at 48-50℃and 1 min at 72℃,and finally,by 1 cycle for 10 rain at 72℃ ,stored at 4 ℃.