开口箭ISSR-PCR实验反应体系条件的优化

Optimization for ISSR-PCR Reaction System of Tupistra chinensis

目的:建立与优化药用植物开口箭ISSR-PCR实验反应体系。方法:以开口箭DNA为材料,分析了Mg2+,dNTP,引物及Taq DNA聚合酶浓度对ISSR-PCR扩增结果的影响。结果:确立了稳定的、可重复的开口箭ISSR最佳反应体系,即在10μL的PCR反应体系中,含1×buffer,2 U Taq DNA聚合酶,2 mmol.L-1MgCl2,0.2 mmol.L-1dNTP,0.75μL引物。结论:从80条简单序列重复区间(ISSR)通用引物中筛选出了16条条带清晰稳定的引物,设置了不同的引物退火温度,为进一步进行开口箭的遗传多样性分析的研究奠定了基础。

Objective： To establish the inter simple squence repeat（ISSR）-PCR reaction system of Tupistra chinensis.Method： The total genome DNA of T.chinensis.was used as the material.The concentration of Mg2 ＋,dNTP,primer and Taq DNA polymerase,which greatly influence ISSR-PCR reaction,were optimized.Result： The optimized reaction conditions was established as follows： in a volume of 10 μL containing 1 × buffer,2 U Taq DNA polymerase,2 mmol.L-1 MgCl2,0.2 mmol.L-1 dNTP,0.75 μL primer.Conclusion： Sixteen primers with stable amplification and rich polymorphism were screened from 80（inter simple sequence repeat） ISSR primers.The optimized annealing temperature for each primer in ISSR-PCR reaction was proposed.This study will be helpful for research on the genetic diversity analysis and germplasm identification of T.chinensis.