不同分离缓冲液对杉木根尖细胞核悬液DNA分辨率的影响  

Effects of Different Isolation Buffers on DNA Resolution in Nucleus Suspension of Root Tip Cell of Cunninghamia lanceolata(Lamb.) Hook.

作　　者：沈捷[1] 徐进[1] 刘光欣[1] 施季森[1] 

机构地区：[1]林木遗传和生物技术省部共建教育部重点实验室、南京林业大学,南京210037

出　　处：《分子植物育种》2015年第1期190-196,共7页Molecular Plant Breeding

基　　金：国家自然基金重点项目(30930077)资助

摘　　要：为制备高纯度的杉木细胞核悬液供BD Influx TM流式细胞分选仪使用,本研究配制了6种细胞核分离缓冲液制备样品,就不同分离缓冲液对DNA分辨效果进行比较。研究结果显示,WPB buffer不能获得有效分辨的峰形,而OTTO＇s buffer表现为最高的分辨率,G0/G1峰的变异系数为4.61%。其余4种细胞核分离缓冲液（Arumuganathan＇s buffer,Galbraith＇s buffer,Lysis buffer LB01和Marie＇s nuclear isolation buffer）的变异系数在5%~8%之间（分别为5.49%,7.30%,5.12%和6.89%）。本研究分别选用了DAPI和PI对细胞核悬液进行染色发现,利用DAPI染料获得的柱状图CV值更小、分辨率更高。研究结果表明,利用Lysis buffer LB01分离缓冲液,结合DAPI染色,可以制备得高纯度的杉木根尖细胞核悬液。这一结果为杉木根尖细胞有丝分裂中期同步化和染色体分选提供实验依据。In order to prepare the high-purity Chinese fir nucleus suspensions for analysis by the BD InfluxTM Cell Sorter, six different buffers were designed to compare the DNA resolution effect of Chinese fir nucleus suspensions. The results showed that WPB buffer was not suitable for isolating nucleus suspensions, while OTTO＇s buffer showed the highest resolution with lower coefficient of variation （CV value was equal to 4.61%） at G0/G1 peaks. Other four buffers （Arumuganathan＇s buffer, Galbraith＇s buffer, Lysis buffer LB01 and Marie＇s nuclear isolation buffer） had the higher variation with coefficient of 5.49%, 7.30%, 5.12% and 6.89%, respectively. Flow karyotype graph showed that the lower coefficient of variation obtained by using DAPI to stain nucleus suspensions. It was confu＇med that the Lysis buffer LB01 with DAPI staining should be applicable to prepare high-purity Chinese fir nucleus suspensions. These results would provide experimental basis for studying on synchronization process of root-tip mitosis cells in Chinese fir as well as on sorting of chromosomes by flow eytometry.

关 键 词：杉木 流式细胞术 细胞核悬液 细胞核分离缓冲液 

分 类 号：S791.27[农业科学—林木遗传育种]