正交设计优化果梅ISSR反应体系

Optimization of an ISSR reaction system of Japanese apricot (Prunus mume) based on orthogonal design

以果梅（Prunus mume Sieb．et Zucc．）品种鸳鸯梅为试材，采用改良的CTAB法提取果梅嫩叶DNA，利用正交设计L16（4^5）探讨Mg^2＋、dNTPs、引物、Taq DNA聚合酶及模板DNA用量对果梅ISSR—PCR反应的影响，正交试验的结果采用直观分析和方差分析相结合。建立了果梅的ISSR—PCR优化反应体系，在20μL反应体系中含2μL 10×Buffer，2．5mmol·L^-2 Mg^2＋，0．2mmol·L^-1 dNTPs，0．32μmol·L^-1引物，20—80ng模板DNA，0．75U Taq DNA聚合酶。在此基础上探讨了引物UBC840的最适退火温度、最佳循环次数及延伸时间，引物UBC840的最适退火温度为50．6℃。应用该优化反应体系，用2个不同引物对19份果梅资源DNA进行ISSR—PCR扩增，结果显示优化的反应体系具有较高的稳定性。

Japanese apricot （Prunus mume Sieb. et Zuec.） genomic DNA, extracted from its fresh young leaves by modified CTAB method Orthogonal design was applied to optimize ISSR-PCR amplification system of Japanese apricot in five factors such as Mg^2＋, dNTPs, primer, Tat/DNA polymerase and template DNA at four levels. The result of PCR was analyzed by intuitive analysis and variance analysis. An optimal reaction system （20 μL） was established, i.e. 2 μL 10× Buffer, 2.5 mmol· L^-1 Mg^2＋, 0.2 mmol·L^- 1 dNTPs, 0.32 μmol· L^-1 primer, 20-80 ng template DNA, 0.75 U Taq DNA polymerase. The annealing temperature ,the number of cycles and the extension time were also investigated. 50.6 ℃ was the most suitable annealing temperature of UBC840 primer. 19 euhivars of Japanese apricot were used to test the stabilization of the optimized reaction system. The results indicated that the optimized reaction system was very stable.