贵州石笔木DNA提取与ISSR-PCR反应体系的建立

Establishment of DNA Extraction and ISSR-PCR Reacting System of Tutcheria kweichowensis

[目的]利用ISSR技术对贵州石笔木的遗传多样性进行分析。[方法]以贵州石笔木种子、新鲜叶片和干燥叶片为试材,采用改良的CTAB法和SDS法提取贵州石笔木基因组DNA,并对提取DNA的浓度和纯度进行测定。通过单因子梯度试验,筛选出优化、稳定的ISSR-PCR反应体系。[结果]利用CTAB法和SDS法从石笔木叶片中提取DNA时,鲜叶和干燥叶片提取的DNA质量和浓度略有差别,CTAB法提取的浓度稍大而蛋白残留稍多,SDS法提取的DNA量稍少但质量最高。而从种子中提取的DNA无论质量还是浓度均较差,但尚可满足ISSR分子标记试验。贵州石笔木的适宜ISSR-PCR反应体系为2μl的10×Buffer,模板DNA 40 ng,dNTP 0.25 mmol/L,Taq酶1 U,引物0.2μmol/L,总体积为20μl。[结论]该ISSR技术体系适用于贵州石笔木遗传多样性分析。

[ Objective ] The research aimed to lay the foundation for the genetic diversity analysis of Tutcheria kweichowensis by ISSR technology. [ Method] The materials of the fresh leaves, dry leaves and seeds of Tutcheria kweichowensis and the improved methods of SDS and CTAB were used for the DNA extraction experiment, and the concentration and purity of the extracted DNA were determined. Through single-factor gradient test, the optimized ISSR-PCR reaction system was screened out. [ Result] When extracting the leaf DNA of Tutcheria kweichowensis with the methods of CTAB and SDS, there were small differences in quality and density between the fresh leaves and dry leaves. There were higher density and more protein residues by CTAB than those by SDS. The quantity of DNA with SDS was a little less than that of CTAB but with the highest quality. Both the quality and density of DNA extracted from seeds were worse, but still qualified for ISSR molecular marking experiment. The appropriate ISSR-PCR reacting conditions were as follows： 20 ul reacting system, 10 x Buffer, 1 U Taq DNA enzyme, 0.2 μmol/L primer, 0.25 mmol/L L dNTP and40 ng template DNA. [ Conclusion] This ISSR reaction system was suitable for the genetic diversity analysis of Tutcheria kweichowensis.