摘要
本文介绍了福建柏Fokieniahodginsii(Dunn)HenryetThomas总DNA的提取介质组成分 ,建立了一个快速、简便且高效提取和鉴定福建柏鲜叶、干叶、种子总DNA的方法。该方法所提DNA的产率分别为 :1 5 0～ 2 5 0 μg/g鲜叶 ,1 0 0～ 1 5 0 μg/g干叶和 1 4 0～ 1 6 0 μg/g种子 ;绝大多数粗提总DNAOD2 6 0 /OD2 80 =1 .80±0 .0 2 ;提取的鲜叶、种子和单种子胚乳总DNA皆为 4 8kb,而干叶总DNA为 5 0Kb ,都适于限制性酶切和RAPD反应 ;一般实验时间为 4h。该方法既不需氯化铯梯度离心和柱层析纯化 ,也不需氯仿 :异戊醇抽提 ,粗提的总DNA样品不经RNase消化即可用于RAPD反应或经RNase消化用于限制性酶切 ,因此 ,具有高产率、高纯度、高质量和快速简便等优点。尤其是它所提取的干叶DNA ,适于限制性酶切和PCR反应 ,解决了取材上的不便。实验中还发现 :( 1 )提取DNA之前去除叶绿素与否 ,对提取福建柏总DNA无明显影响。 ( 2 )只有去除RNA的福建柏总DNA样品 ,才能被限制性内切酶完全切开。 ( 3)RAPD模板中含RNA及一定量的蛋白质 ,均不影响扩增效果。 ( 4 )同一个体的鲜叶、干叶、种子总DNA样品 ,皆可得到完全一致的扩增产物。
The recent studies of Fokienia hodginsii (Dunn) Henry et Thomas, which is a rare and endangered tree species, show that it can play an important role in the sustainable development of the forest land. So it has been listed as one of the most important tree species in afforestation by the Forestry Ministry of China. A fast, simple and convenient method is established to isolate and characterize the total DNA in fresh leaves, dry leaves and seeds of Fokienia hodginsii . The production rate of these isolated DNA are 150～250 μg DNA per gram of fresh leaves, 100～150 μg DNA per gram of dry leaves, 140～160 μg DNA per gram of seeds respectively, and the rough total DNA OD 260 /OD 280 =1.80±0.02. The total DNA from fresh leaves, seeds and one seed's endosperm is 48 kb respectively, but that from dry leaves is 50 kb. Therefore, these DNA are all suitable for digesting with restriction enzyme and RAPD analysis, generally the experiment either time is only 4?h. It is not necessary to carry on cesium chloride gradient contrifugation and column chromatography or abstraction with chloroform. The rough total DNA can be used in RAPD analysis without RNase digestion, and in digesting with restriction enzyme after being digested by RNase as well. Consequently, it is easy to obtain high yields, high purity and high quality of total DNA in 4?h. Especially, the total DNA in dry leaves obtained by this method has a higher level in digesting with restriction enzyme and PCR analysis, thus it solves the inconvenience of drawing materials. During experiment, some useful results have also been found: (1)It has no obvious influence on the isolation of total DNA from Fokienia hodginsii whether eliminating chlorophyll before isolation or not. (2) The total DNA can be digested completely only its RNA is eliminated. (3) The RAPD template that has RNA and a certain amount of protein shows no effect on the result of RAPD. (4) DNA templates extracted from fresh leaves, dry leaves or seeds of an individual produce the same RAPD result.
出处
《林业科学》
EI
CAS
CSCD
北大核心
1999年第5期51-56,共6页
Scientia Silvae Sinicae
基金
福建省自然科学基金!(C9810 0 13 )
福建省教委资助!(JA970 43 )
关键词
福建柏
总DNA
提取
提取介质
RAPD
鉴定
Fokieniahodginsii , Isolation of total DNA, Extraction medium, Digestion with restriction enzyme, RAPD
