油茶DNA提取及RAPD分析最佳反应体系的建立

DNA Extraction from Camellia oleifera and the Establishment of the Optimum Reaction System of RAPD Analysis

[目的]为建立油茶RAPD分析的最佳反应体系。[方法]分别用改良SDS法和CTAB法从12个油茶品种的新鲜幼叶中提取基因组DNA。通过测定OD260和OD280来比较两种方法提取DNA的纯度。建立油茶的RAPD反应程序,通过优化反应条件获得油茶RAPD分析的最佳反应体系。[结果]CTAB法提取的油茶基因组DNA纯度高于SDS法。油茶RAPD分析的最佳反应体系为:1.0 UTaq DNA聚合酶、1.5 mmol/L MgCl22、00μmol/L dNTP1、0 pmol/L随机引物2、0 ng DNA模板2、μl 10×PCR buffer,加重蒸水至20μl。油茶的RAPD反应程序为:95℃预变性3 min;94℃变性1 min,40℃退火1 min,72℃延伸2 min,40循环;72℃延伸7 min。[结论]该研究所用DNA纯度较高,反应条件控制严格,试验中扩增稳定性较好,是适合油茶RAPD分析的最佳反应体系。

[Objective] The research aimed to establish the optimum reaction system of RAPD analysis for Camellia oleifera. [Method] Genomic DNA was extracted from fresh tender leaves in 12 species of C. oleifera by using the improved SDS method and CATB respectively. The purity of DNA extracted by 2 kinds of methods was compared by determining the values of OD260 and OD280. The RAPD reaction procedure for C. oleifera was established, and the optimum reaction system of RAPD analysis for C. oleifera was obtained through optimizing the reaction conditions. [ Result ] The purity of genomic DNA extracted from C. oleifera by CrAB method was higher than that by SDS method. The optimum reaction system of RAPD analysis for C. oleifera was as follows： 1.0 U Taq DNA polymerase, 1.5 mmol/L MgCl2, 200 μmol/L dNTP, 10 pmol/L random primers,20 ng template DNA,2μl 10 × PCR buffer, adding the redistilled water till 20 μl. The RAPD reaction procedure for C. oleifera was as follows： predenaturation at 95 ℃ for 3 min; denaturation at 94 ℃for 1 min, annealing at 40 ℃ for 1 rain, extension at 72 ℃ for 2 min, 40 cycles and extension at 72℃ for 7 min, [ Conclusion] The DNA used in this research had high purity, the reaction conditions were strictly controlled and the amplification stability in the test was better, which was the optimum reaction system of RAPD analysis for C. oleifera.