赤皮青冈ISSR-PCR反应体系的建立与优化

摘要

In order to establish the optimal system of ISSR-PCR for Cyclobalanopsis gilva, with the genomic DNA of C.gilva leaves as material, single factor test and orthogonal experiment design were used to systematically test six parameters of ISSR-PCR which include template DNA, Mg2+, primers, dNTPs concentration, dose of Taq DNA polymerase and annealing temperature.Integrated analysis implies that the optimal reaction system of ISSR (20μL) is:20 ng template DNA, 2.5 mmoL/L Mg2+, 0.2 mmoL/L dNTPs, 0.2 µmoL/L primers and 1.0 U Taq DNA polymerase, if UBC808 as the primers in ISSR-PCR that the optimized annealing temperature is 59.3℃. The establishment of the optimal system lays an important foundation for using ISSR technology in research C.gilva genetic diversity in future.%为建立适宜于赤皮青冈ISSR分析的扩增体系，以赤皮青冈叶片基因组DNA为材料，采用单因素和正交设计相结合的方法系统地测试模板DNA、Mg2+、引物、磷酸碱基脱氧核苷（dNTPs）浓度，Taq DNA聚合酶用量和退火温度这6个因素对ISSR-PCR反应结果的影响。综合分析表明：优化后的最佳反应体系为20µL反应总体系中，含20 ng模板DNA，2.5 mmoL/L Mg2+，0.2 mmoL/L dNTPs，1.0 U Taq DNA聚合酶，0.2µmoL/L引物；UBC 808号作引物最佳退火温度为59.3℃。这一优化体系的建立为进一步开展赤皮青冈遗传多样性的ISSR分析奠定了基础。