思茅松大配子体DNA的提取及ISSR反应体系的建立

为建立思茅松大配子体ISSR分子标记的反应体系,以思茅松种子样本作为供试材料,比较不同思茅松胚乳DNA提取方法的效果,通过紫外分光度计、DNA琼脂糖电泳和PCR扩增分别予以检测并比较。结果表明SDS法优于CTAB法。同时,通过单因子分析法对ISSR-PCR反应体系中的模板DNA用量,Taq DNA聚合酶用量,Mg2＋浓度,dNTP浓度、引物浓度等主要影响因子进行优化,确立适用于思茅松的ISSR分子标记反应体系。对该体系进行稳定性检验,结果表明该反应体系适合思茅松ISSR-PCR扩增。

The DNA Extraction in Macrogametocyte of Pinus kesiya var.langbianensis and Establishment of ISSR Reaction System

Different methods of DNA extraction were compared by using the ultraviolet spectrophotometer,DNA agarose electrophoresis and PCR amplification,in order to establish the ISSR reaction system of the macrogametocytes of Pinus kesiya var.langbianensis,in which the seeds were used as materials.The results showed that the method of SDS was better than CTAB,and meanwhile,the ISSR reaction system was established by the analysis of the main factors such as the usage of template DNA and Taq DNA polymerase,the concentration of Mg2+,dNTP and primer.The stability of the ISSR reaction system was tested and the result showed that this reaction system was suited to the ISSR-PCR amplification of Pinus kesiya var.langbianensis.