CAJ | 학술논문

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( Betula alnoides )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.
종부함다당적완요식물류군제취여순화DNA시허다연구영역례여거군생물학、생물다양성、분자표기보조육충연구보편우도적난제.이서남화( Betula alnoides )위례발전료일투개진적방안,유효지종저충완요식물적간협화선협중제비료DNA.차방안포괄3개관건보취:수선종식물세포균장중용불함CTAB적완충액세거대부분다당화기타차생물질;재제취개질중채용3% CTAB이불시통상용적2% CTAB;장상용적고염거당적순화조작제전도용이병순침정DNA지전진행.종서남화제취적DNA이성공지용우RAPD-PCR확증화한제성매절.저개간단、경제화가고적개진방안야괄용우허다기타적완요식물류군.
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.