濒危植物格氏栲叶片基因组DNA提取方法研究

Study on Extraction of Genomic DNA from Castanopsis kawakamii Leaves

格氏栲植物体内酚类、多糖等次生物质含量较高,严重影响提取其基因组DNA的产量和质量。通过对格氏栲叶片DNA几种提取方法获得的DNA质量分析,结果表明,改良CTAB法是最为理想的提取方法。加入质量分数为10%的PVPP粉与材料充分研磨,在提取缓冲液中加入体积分数为3%的β-巯基乙醇,能有效地去除材料中的多酚类物质;用氯仿/异戊醇(241∶)抽提裂解液2次所获得的上清,加入1/5倍体积的7.5mol/L NH4Ac,再用异丙醇沉淀DNA,可以有效去除多糖的干扰,所提取的DNA纯度较高,可用于PCR扩增。

The content of phenolic compound, polysaccharides and other secondary metabolites is high in Castanopsis kawakamii, which influence the yield and quality of genome DNA while being extracted. Based on the analysis of several isolation methods for genomic DNA of Castanopsis kawakomii, it was found that the improved CTAB method was the best.The results showed that polyphenolics were effectively removed from materials by blendering 10% Poly Vinyl Poly Pyrrilodone （PVPP） to the ground materials under liquid nitrogen in mortar and highering concentration of 13 - mercaptoethanol in extracting buffer.to 3% ;the polysaccharides were effectively removed by adding 1/5 volume 7.5mol/L NH4Ac and absolute ethanol to the blundering aqueous phase after successively extracting lysis solution twice with 24 ： 1 chloroform： isoamyl alcohol; the purer DNA sample was obtained, and which was suitable for PCR analysis.