Preparation of Total DNA from "Recalcitrant Plant Taxa

顽拗植物类群的总DNA制备(英文)

Contamination problems on DNA isolation from 'recalcitrant plant taxa' which is rich in polysaccharides have been commonly encountered in a wide range of research fields such as plant population biology, biodiversity, and molecular marker-assisted breeding. Here we present an improved protocol to extract DNA efficiently from dry or fresh leaves of a 'recalcitrant plant taxa', Betula alnoides Buch. Ham. ex D. Don in which three key steps are involved: 1) washing out most of polysaccharides and other secondary compounds with CTAB-free buffer from homogenate; 2) adoption of 3% CTAB rather than 2% CTAB in the exaction medium; and 3) using of high concentration of salt prior to DNA precipitation with isopropanol to remove residual polysaccharides. The isolated DNA has been proved suitable for RAPD-PCR amplification and restriction digestion. This modified procedure is simple, inexpensive and reliable, and is also applicable to many other plant taxa with high polysaccharides.

从富含多糖的顽拗植物类群提取与纯化DNA是许多研究领域例如居群生物学、生物多样性、分子标记辅助育种研究普遍遇到的难题。以西南桦 (Betulaalnoides)为例发展了一套改进的方案 ,有效地从这种顽拗植物的干叶和鲜叶中制备了DNA。此方案包括 3个关键步骤 :首先从植物细胞匀浆中用不含CTAB的缓冲液洗去大部分多糖和其他次生物质 ;在提取介质中采用 3%CTAB而不是通常用的 2 %CTAB ;将常用的高盐去糖的纯化操作提前到用异丙醇沉淀DNA之前进行。从西南桦提取的DNA已成功地用于RAPD_PCR扩增和限制性酶切。这个简单、经济和可靠的改进方案也适用于许多其他的顽拗植物类群。