辽细辛和北马兜铃ISSR-PCR的引物筛选及反应体系的建立与正交优化

Primer Screening of Aristolochicaceae-Liao Asarum and Aristolochia mansoni by ISSR-PCR and Establishment and Orthogonal Optimization of Reaction System

目的探寻适合细辛和马兜铃品种鉴定的ISSR引物。方法利用一个品种对全部100条ISSR引物设置退火温度进行梯度实验,筛选出重复性好、条带清晰的引物5条,从理论上讲,这些筛选的引物可以实现对现有辽细辛和北马兜铃品种的鉴定。马兜铃属ISSR-PCR的引物筛选,利用正交试验设计的方法,从Taq DNA聚合酶浓度、Mg^(2+)浓度、dNTP浓度、引物浓度,这4种因素3个水平,对马兜铃属ISSR-PCR反应体系进行优化分析,并在此基础上对模板DNA浓度、PCR反应过程中的退火温度进行梯度检测。结果从样品中筛选出优选引物,细辛UBC815、UBC826、UBC862、UBC868、UBC888;马兜铃UBC825、UBC827、UBC836、UBC846、UBC848。反应体系的稳定性进行检测结果均稳定可靠。结论得到马兜铃的最佳组合,即20μL ISSR-PCR反应体系中含0.25 Lmol/L引物、1.5 mmol/L Mg^(2+)、1×PCR buffer、150 Lmol/L dNTP和0.5 UTaqDNA聚合酶。PCR反应条件为94℃预变性5 min,以30个循环94℃变性30 s,50℃退火1 min,72℃延伸1 min,最后以72℃延伸10 min。得到细辛的最佳组合,20μL ISSR-PCR反应体系中含0.50 Lmol/L引物、1.5 mmol/L Mg^(2+)、2×PCR buffer、250 Lmol/L dNTP和2.0 UTaqDNA聚合酶;同时得到最佳模板DNA浓度为10 ng,最佳退火温度为50℃。

Objective To explore the ISSR primers for identification of varieties and Aristolochia asarum. Methods The annealing gradient experiment for all 100 ISSR primers were screened using a variety of high polymorphism primers, 5 primers with good repeatability, theoretically, these primers screening can achieve identification of existing species of Aristolochia contorta and asarum. Primer screening of Aristoloehia ISSR-PCR, using the method of orthogonal experimental design, the primer concentration, Taq DNA polymerase concentration, Mg^2＋ concentration and dNTP concentration of 4 factors and 3 levels to optimize the analysis of Aristolochia ISSR-PCR reaction system, and on the basis of template DNA concentration, annealing temperature of PCR reaction in the process of gradient detection. Results Preferably selected primers from samples, UBC815, UBC826, UBC862, UBC868, UBC825, UBC827, UBC888; aristolochic UBC836, UBC846, UBC848. The stability of the reaction system is stable and reliable. Conclusion he optimal combination of aristolochic acid, namely 20μL ISSR-PCR reaction system containing 1 × PCR buffer, 150Lmol / L dNTP, 0.25Lmol / L primer, 1.5mmol / L Mg^2＋ and 0.5UaqDNA polymerase. The PCR reaction conditions were d, enaturation at 94%; for 5 rain, denaturation at 94% for 30s, annealing at 50 ℃ for 1 min, extension at 72 ℃ for 1 rain and extension at 72 ℃ for 10 min. The optimal DNA concentration was 20ng and the optimal DNA concentration was 20ng. The optimal conditions were as follows： 20μ L ISSR-PCR reaction system containing 2 × PCR buffer, 250Lmol / L dNTP, 0.50Lmol / L primer, 1.5mmol / L Mg^2＋ and 2.0UTaqDNA polymerase; And the optimum template DNA concentration was 10 ng ,The annealing temperature was 50 ℃.