油松cDNA SRAP-PCR反应体系的建立与优化

Establishment and Optimization of cDNA-SRAP PCR Reaction System in Chinese Pine

为了建立油松cDNA SRAP-PCR反应体系,利用正交设计L16(45)对PCR反应体系的Taq酶、模板cDNA、引物、Mg^(2+)、d NTP这五个因素在四个水平上进行优化。使用SPSS软件对PCR结果结合方差分析和直观分析。结果表明:不同因素水平的变化对SRAP-PCR反应的影响大小依次为d NTP>模板c DNA>Taq酶>引物>Mg^(2+)。筛选各因素最佳水平,油松c DNA SRAP-PCR最佳反应体系(20μL):Taq酶2 U,c DNA模板用量80~120 ng,引物浓度0.4μmol/L,Mg2+浓度1.5 mmol/L,d NTP浓度0.2 mmol/L。这一优化体系的建立有助于今后利用cDNA-SRAP技术对油松进行基因表达差异的分析。

In order to establish the SRAP-PCR system on Chinese pine(Pinus tabulaeformis Carr), the PCR system was optimized in those five factors, Taq DNA polymerase, Template, primer, Mg^(2+) and d NTP, at four level by using orthogonal design L16(45). Software SPSS was used in variance analysis and intuitive analysis on the results of PCR. The results indicated that the effects of the different factors on the SRAP-PCR system in turn of the following order: d NTP >c DNA template >Taq DNA polymerase >primer>Mg^(2+). Screening the optimal level of factors. the SRAP-PCR system for Chinese pine contains 2.0 U Taq DNA polymerase, 80~120 ng cDNA template,0.4 μmol/L primer, 1.5 mmol/L Mg^(2+), 0.2 mmol/L d NTP in total 20 μL. This optimized system for Chinese pine would be useful in differential gene expression analysis by using the cDNA-SRAP technique.