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

关键词:  永瓣藤  珍稀濒危植物  DNA提取  ISSR

Key words:  Monimopetalum chinense  rare and endangered plant  DNA extraction  ISSR

摘要:

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20 μL反应体系中,含20 ng模板DNA、2.375 mmol/L Mg²⁺、0.15 mmol/L dNTPs、1.5 U Taq DNA聚合酶、225 nmol/L随机引物; 扩增程序为:94 ℃预变性5 min; 然后94 ℃ 15 s、48～50 ℃ 45 s、72 ℃ 1 min,35个循环,最后72 ℃延伸10 min,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。

Abstract:

In the present study,DNA of Monimopetalum 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 Mg²⁺,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 min at 72 ℃,stored at 4 ℃.