分离缓冲液对水稻细胞核悬液DNA分辨率效应的比较  

Comparison on the effects of different isolation buffer on the DNA resolution of the cell nucleus suspensions from rice

作　　者：金亮[1] 张宪银[1] 薛庆中[1] 

机构地区：[1]浙江大学农业与生物技术学院农学系,浙江杭州310029

出　　处：《浙江农业学报》2007年第2期93-96,共4页Acta Agriculturae Zhejiangensis

基　　金：国家自然科学基金(30571146);浙江省水稻攻关项目(0406)

摘　　要：为制备高纯度水稻细胞核悬液供FACSCalibur流式细胞仪使用,选用6种细胞核分离缓冲液制备样品,对其DNA分辨率效果进行比较,Otto buffers和Marie’s nuclearisolation buffer表现分辨率较高,细胞G0/1峰产生的变异系数较低(CV分别为3.57%和3.41%);其中Otto buffers的组分较Marie’s nuclear isolation buffer简单、成本低,且能在正常室温条件下分析和保存。虽然在细胞核悬液中Tris-MgCl2buffer显示最低的CV值(3.19%),但产生较多的背景碎片。而其余3种缓冲液(Lysis buffer LB01,Galbraith’s buffer和Arumuganathan’sbuffer)的变异系数均较高(分别为4.75%,6.28%和6.73%)。结果表明采用Otto buffers分离缓冲液制备细胞核悬浮液,能实现苗期对水稻花粉植株的不同倍性(单倍体、二倍体、三倍体等)快速鉴定。In order to prepare the high-purity rice nucleus suspensions analyzed in the FACSCalibur Flow Cytometer, six different buffer were compared for the resolution effect of samples DNA. Otto buffers and Marie＇ s buffer showed the higher resolution with lower coefficient of variation （CV values were equal to 3.57% and 3.41% respectively） at G0/1 peaks. Otto buffers is simpler in components, and lower in cost than Marie＇s buffer, also it can be analyzed and conserved under normal room temperature conditions. Tris-MgCl2 buffer exhibited the lowest CV value （3.19%）, but produced more background debris in all the nuclus suspensions. Other three kind of buffer（Lysis buffer LB01, Galbraith＇ s buffer and Arumuganathan＇ s buffer） have the higher coefficients of variation （4.75%, 〉 6.28% and 〉 6.73% respectively）. By the Otto buffer in the preparation of cell nucleus suspension, we have identificated fast and correct their different ploidy level （haploids, diploids, and triploids） from leaves of pollen plants at the seedling stage.

关 键 词：水稻 流式细胞术 核悬液 细胞核分离缓冲液 

分 类 号：S511.035[农业科学—作物学]