峨眉金线莲ISSR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System of Anoectochilus emeiensis

摘要: 利用5因素4水平的正交实验探讨了各因素的不同水平对峨眉金线莲ISSR-PCR反应结果的影响，建立了稳定且重复性好的峨眉金线莲ISSR-PCR反应体系，即在25 μL体系中含：40 ng DNA模板、0.4 μmol·L⁻¹引物、0.4 mmol·L⁻¹ dNTPs、2.0 mmol·L⁻¹ Mg²⁺、0.8 U Taq DNA 聚合酶和2.0 μL 10× Buffer。同时为了确定最适Tm（退火温度），进行了梯度PCR试验筛选，最终优化和确立了适合峨眉金线莲的ISSR-PCR实验反应体系。

Abstract: The effects of different levels of factors on ISSR-PCR reaction results of Anoectochilus omeiensis were discussed by orthogonal design with 5 factors and 4 levels, and a stable and reproducible ISSR-PCR reaction system of Anoectochilus omeiensis was established. That is, the 25 μL system contained 40ng DNA template, 0.4μmol/L primer, 0.4 mmol/L dNTPs, 0.8 U Taq DNA polymerse, 2 mmol/L Mg²⁺, and 2.0 μL 10×Buffer μL in 25 μL reaction volume. At the same time, in order to determine the optimum Tm （annealing temperature）, gradient PCR test was carried out, and finally ISSR-PCR experimental reaction system suitable for Anoectochilus omeiensis was optimized and established.