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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 and optimize the ISSR-PCR experiment system for the opening of medicinal plants. Methods: The effect of Mg2 +, dNTP, primer and Taq DNA polymerase on the amplification results of ISSR-PCR was analyzed by using open arrow DNA. Results: A stable and repeatable ISSR system with open arrows was established. In a 10 μL PCR reaction system 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 clear and stable primers were screened from 80 Simple Sequence Repeats (ISSR) universal primers and different primer annealing temperatures were set up, which laid the foundation for the further study on the genetic diversity of open arrows .