目的:建立蕺菜rDNA ITS扩增反应的优化体系。方法:以蕺菜DNA为模板,采用单因素试验设计,对影响蕺菜rDNAITS区扩增的重要参数进行了优化试验。结果:最优蕺菜ITS-PCR的反应体系为25μl反应体系中含Taq酶0.75U、Mg^2+2.5mmol/L、dNTP0.15mmol/L、引物对0.6μmol/L、模板DNA10ng。扩增程序为在94℃下预变性4min,然后进行36个循环(94℃变性45s,60℃退火50s,72℃延伸90s),最后在72℃延伸8min。结论:这一体系的建立为利用蕺菜rDNAITS区研究蕺菜种质资源的系统进化提供了标准化程序。
Objective:we optimized PCR conditions for amplifying rDNA ITS from Houttuynia Thunb. Method and Result:The results indicated that the best ITS-PCR reaction system should follow the conditions as follows: Taq DNA polymorase0.75U, Mg^2+ 2.5mmol/L, dNTP 0.15mmol/L, primers 0.6μmol/L, DNA 10ng. PCR procedures are described as follows: 94℃ for 4min, followed by 36 cycles (94℃ for 45s, 60℃ for 50s, 72℃ for 90s),finally extended at 72℃ for 8min. Conclusion:The result provided a standard ITS-PCR program for the analysis of phylogenic and evolutionary of Houttuynia Thunb.