分子植物育种
(
网络版
), 2010
年
,
第
8
卷
,
第
7
篇
Fenzi Zhiwu Yuzhong (Online), 2010, Vol.8, No.7
http://mpb.
5th
.sophia
p
ublisher.com
共
5
页,第
1
页
技术改进
Upgraded Technology
高羊茅
RNA
提取方法的比较研究
王康
1
,
李恩杰
1
,
董洁
2
,
周禾
1*
1
中国农业大学动物科技学院草业科学系
,
北京
, 100193
2
中国农业科学院北京畜牧兽医研究所
,
北京
, 100193
通讯作者
:
zhouhe@cau.edu.cn;
作者
分子植物育种
, 2010
年
,
第
8
卷
,
第
7
篇
DOI: 10.5376/mpb.cn.2010.08.0007
收稿日期:
2010
年
8
月
15
日
接受日期:
2010
年
9
月
14
日
发表日期:
2010
年
10
月
28
日
这是一篇开放阅取的论文,其论文发布和传播接受《
Creative Commons Attribution License
》所有条款。只要对本原作有恰当的引用,版权所有
人允许和同意第三方无条件的使用、传播以及任何媒介的复制或再制作。
建议的引用格式如下:
王康等
, 2010,
高羊茅
RNA
提取方法的比较研究
,
分子植物育种
Vol.8 No.7 (DOI: 10.5376/mpb.cn.2010.08.0007)
摘要
高羊茅
(
Festuca arundinacea
Schreb.)
是一种重要的牧草和草坪兼用型草种,在国内外农牧业和草坪业中占据重要的地
位。高质量
RNA
的提取是高羊茅分子生物学研究的必要前提。本实验以高羊茅叶片为材料,采用常用的
2
种方法对其总
RNA
进行提取,同时对前人的
RNA
提取方法进行改良,寻求最佳的高羊茅总
RNA
提取方法。通过比较发现,
Trizol
法价格昂贵
不适合大量植物组织提取,传统的异硫氰酸胍法所提取的总
RNA
有蛋白质污染且
RNA
有所降解,改良后的异硫氰酸胍法
所获得的总
RNA
经琼脂糖凝胶电泳检测,可清晰的看到
28S rRNA
、
18S rRNA
和
5S rRNA 3
条带,且
28S rRNA
的亮度大
于
18S rRNA
的亮度,
OD260/OD280
比值介于
1.8~2.0
之间,
OD260/OD230
比值大于
2.0
,表明该方法提取的
RNA
完整性
好,纯度高。通过
cDNA-AFLP
分析方法的检测,可得到条带清晰、多态性丰富的图谱,说明该方法提取的总
RNA
可直接
用于后续的分子生物学研究中,为进一步的高羊茅分子生物学研究打下良好基础。
关键词
高羊茅
;
总
RNA
提取
;
改进的异硫氰酸胍法
Comparison and improvement of high-quality RNA extraction methods from
Tall fescue
Wang Kang
1
, Li En-jie
1
, Dong Jie
2
, Zhou He
1*
1 Department of Grassland Science, College of Animal Science and Technology, China Agricultural University, Beijing, 100193
2 Institute of animal science, CAAS, Beijing, 100193
Corresponding author, zhouhe@cau.edu.cn;
Authors
This article was first published in Molecular Plant Breeding, and was distributed in Chinese under the terms of the Creative Commons Attribution License.
Abstract
Tall fescue (
Festuca arundinacea
Schreb.), an important lawn and pasture grass in agriculture, occupy an important
position in animal husbandry and lawn industry. The isolation of high quality RNA was a precondition of Molecular biology research.
In this experiment, we adopt two common approaches as well as the improved isothiocyanate method for total RNA extraction by
using the leaves of tall fescue as the material in order to seek the optimum total RNA extraction method of tall fescue. The results
showed that, Trizol method with high reliability was the simplest approach, but it’s costly. And there was protein significantly
pollution and degradation of the RNA by the isothiocyanate method. By employing improved isothiocyanate method, we found that
there were three bright bands in agarose gel electrophoresis, 28S rRNA, 18S rRNA and 5S rRNA. The band of 28S rRNA was
brighter than that of 18S rRNA, and the value of OD
260
/OD
280
was 1.8 to 2.0. Clear bands and high polymorphisms have been got by
cDNA-AFLP analysis. These results indicated that the RNA isolated by improved isothiocyanate method had a good integrity and
high purity, and could be used for the later molecular researches.
Keywords
Tall fescue; RNA extraction; Improved isothiocyanate method
研究背景
高羊茅
(
Festuca arundinacea
Schreb.)
为禾本科
羊茅属植物,多年生草本,丛生型,须根发达,适
应性广,并具有耐寒、耐践踏、抗病性强、竞争力