青海云杉ISSR-PCR反应体系的建立与优化

[目的]确定适应青海云杉ISSR-PCR反应的最佳反应体系。[方法]以青海云杉（Picea crassifolia kom.）为材料，对影响其ISSR—PCR扩增结果的各因素（Mg^2＋、dNTPs、TaqDNA聚合酶、模板DNA、引物、退火温度）进行筛选和优化。[结果]青海云杉ISSR—PCR最佳反应体系为在20山反应体系中包含1μl 10×buffer，1．5mmol／L的Mg^2＋ 0．2mmol／L的dNTPs，TaqDNA聚合酶1．0U，模板DNA 40ng，引物0．6μmol／L。对青海云杉ISSR-PCR最佳反应体系的退火温度进行梯度试验发现，引物UBC818的最适退火温度为54．2℃，且最适退火温度因引物而异。[结论]该优化ISSR．PCR反应体系的建立，为今后利用ISSR技术进行青海云杉种质资源分类、遗传图谱构建和基因定位奠定了技术基础。

Establishment and Optimization of ISSR-PCR Reaction System for Picea crassifolia kom.

[Objective]The experiment aimed to determine the optimum ISSR-PCR reaction system of Picea crassifolia kom.. [Method]Picea crassifolia kom. was used as material to select and optimize influencing factors of ISSR-PCR such as Mg2+, dNTPs, TaqDNA polymerase, template DNA, primers, annealing temperature. [Result]The optimum ISSR-PCR reaction system in 20 μl reaction system was consisted of 1 μl 10×buffer, 1.5 mmol/L Mg2+, 0.2 mmol/L dNTPs, 1.0 U TaqDNA polymerase, 40 ng template DNA, 0.6 μmol/L primers. According to gradient test of annealing temperature in optimum ISSR-PCR reaction system of Picea crassifolia kom., it was found that the optimum annealing temperature of UBC818 was 54.2 ℃ and the annealing temperature was different for different primers.[Conclusion]The construction of ISSR-PCR reaction system provided technical basis for classification of germplasm resources, construction of genetic map, gene mapping of Picea crassifolia kom. through using ISSR technology.