摘要
以十字花科植物芸芥 Eruca sativa 、白菜型油菜 Brassica rapa 和甘蓝型油菜 Brassica napus :武39- 1、武4 3- 1、武5 0 - 2为试材,在蔗糖衬垫法分离纯化m t DNA方法优点的基础上,通过4组不同离心力配比,采用差速离心法、改变缓冲液A的成分以及增加缓冲液的用量,建立了一种简便提取十字花科植物mt DNA方法.研究结果表明:分离细胞破碎组织的离心力、分离线粒体的离心力以及纯化线粒体时的离心力分别为10 0 0 g、2 0 0 0 0 g、180 0 0 g时可分离到高产量的线粒体,且在缓冲液A 蔗糖0 .5 m ol·L- 1 ,Tris- Cl5 0 mm ol·L- 1 ,EDTA5 m mol·L- 1 ,BSA 0 .1% ,PVP 0 .5 % ,0 .1%β-巯基乙醇,p H=7.5 中加入0 .5 % PVP可提高mt DNA的产量,经DNA电泳检测、RAPD扩增证明此方法得到的mt DNA可完全达到RAPD标记和其它分子生物学研究的要求.
Wu39-1(Eruca sativa),Wu43-1(Brassica rapa) and Wu50-2 (Brassica napus) were used to develop a handy extraction method of mtDNA in crucifers on the basis of the sucrose-bedding method of mtDNA extraction and by means of four combinations of different centrifugal forces,differential centrifugation,and composition adjustment and dosage increase of buffer solution A.The results demonstrated that with the centrifugal forces to isolate broken cell tissues and mitochondria,and to purify mitochondria at 1 000 g, 20 000 g and 18 000 g,respectively,a high yield of mtDNA could be achieved and the addition of 0.5% PVP to buffer solution A (0.5 mol·L -1 sucrose,50 mmol·L -1 Tris-Cl,5 mmol·L -1 EDTA,0.1% BSA,0.5% PVP,0.1% β-mercaptoethanol,and pH=7.5) could raise the yield of mtDNA;The electrophoresis and RAPD amplification of the mtDNA obtained by the method proved that it could meet the requirements of RAPD marker and other researches in molecular biology.
出处
《西北植物学报》
CAS
CSCD
北大核心
2005年第6期1137-1142,共6页
Acta Botanica Boreali-Occidentalia Sinica
基金
甘肃省自然科学基金项目 ZS991-A2 1-0 43 -N
ZS0 3 1-A2 5 -0 41-D 资助
