紫丁香蘑ISSR-PCR反应体系的正交优化与建立

Optimization and Establishment of Inter-simple Sequence Repeat PCR Rreaction System for Lepista nuda

目的:为了获得紫丁香蘑的最佳ISSR-PCR反应体系。方法:采用正交实验方法 L16(45),对PCR反应的5个重要因素,TaqDNA聚合酶、Mg2+、模板DNA、dNTP以及引物的浓度在4个水平进行优化。结果:得到最佳的反应体系(25μL),包括1.5U TaqDNA聚合酶、2.5 mmol/L Mg2+、0.2 mmol/L dNTP、0.15μmol/L引物、160 ng模板DNA,最佳反应循环数为37。通过极差分析,对紫丁香蘑ISSR-PCR反应的影响程度从大到小依次为:TaqDNA聚合酶、dNTP、模板DNA、Mg2+、引物。使用该优化体系从23个ISSR引物中筛选出6个引物使不同地区的紫丁香蘑具有多态性。结论:研究结果为进一步采用ISSR分子标记方法研究紫丁香蘑的遗传多样性提供了研究基础。

Objective： In order to obtain the optimal ISSR - PCR amplification system of Lepista nuda, an orthogonal design experiment was proceeded in this paper. Method： The 5 factors （Taq DNA enzyme, Mg2 ＋ , template DNA, dNTP, primers） with 4 levels Ll6 （45 ） was con- ducted. Result：The optimal reaction system：25 μL system,Taq DNA enzyme 1.5 U, Mg2＋ 2. 5 mmol/L, template DNA 160 ng, dNTP 0. 2 mmol/L,primer 0. 15 μmol/L. The optimal response procedures need 37 cycles for PCR. The results of range analysis showed that the or- der of the five factors influence degree to ISSR, i. e. Taq DNA polymerase, dNTP, template DNA, Mg2 ＋ , primers. Based on the optimized system,we selected 6 primers,which possessed polymorphism for the different sources Lepista nuda, among 23 ISSR primers. Conclusion： The results of this paper provided the research foundation for the further study of genetic diversity of Lepista nuda.