适用于流式细胞术的三角褐指藻核DNA染色方法  

Flow cytometric analysis of nuclear DNA stained with SYBR Green I in Phaeodactylum tricornutum

作　　者：张志霞[1] 高莉[1] 刘倩倩 曹素娟 兰利琼[1] 卿人韦[1] ZHANG Zhixia;GAO Li;LIU Qianqian;CAO Sujuan;LAN Liqiong;QING Renwei(College of Life Science,Sichuan University,Chengdu 610064,China)

机构地区：[1]四川大学生命科学学院,成都610064

出　　处：《应用与环境生物学报》2018年第6期1270-1274,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(40976092);四川省科技厅项目(2014JY0171)资助~~

摘　　要：为制备可用于配置有488 nm激发器的流式细胞仪检测核DNA含量的三角褐指藻细胞,取对数生长期的三角褐指藻细胞,分别采用70%乙醇、1%戊二醛、1%甲醛溶液进行细胞固定,经RNase A消化后的样品用碘化丙啶(PI)染色,用SYBR-green I对未经RNase A消化的细胞染色,在荧光显微镜下观察细胞染色情况,用流式细胞仪检测细胞核DNA含量.结果显示:在荧光显微镜下观察到,3种不同固定液固定的细胞均表现为SYBR-green I只对藻细胞核染色,PI对于藻的细胞核与细胞质均有着色.流式结果的DNA直方图显示,SYBR-green I染色的细胞的DNA直方图有明显的G1与G2/M峰,拟合度(RCS)在3左右,变异系数(CV)在8.6%左右.而PI染色细胞的DNA直方图表现为变异系数值偏大,拟合度较低,没有明显的G1与G2/M峰.本研究表明,SYBR-green I是一种无需对细胞进行RNase A处理,可应用于配置有488 nm激光器流式细胞仪的核酸染料,可为研究在整个细胞周期的调控机制奠定基础,同时也为其他浮游植物细胞核的染色提供参考.This study aimed to investigate a suitable method to stain the nuclei of Phaeodactylum tricornutum with a flow cytometer equipped with a 488-nm laser to detect the DNA. Exponentially growing cells were fixed using a 70% ethanol, a 1% glutaraldehyde and a 1% formaldehyde solution, respectively. The samples were treated with RNase A and were then stained with propidium iodide. Samples not treated with RNase A were stained with SYBR-green I. All these samples were observed under a fluorescence microscope and the content of the nuclear DNA was quantitated by flow cytometry. Specific fluoresce spots indicate that fluorescence was detected only in the nuclei of cells stained with SYBR-green I and non-RNase A treated, using fluorescence microscopy. The result of flow cytometry showed that the DNA histogram of cells stained with SYBR-green I had distinct G1 and G2/M peaks, with a fit degree (RCS) of about 3, and a coefficient of variation (CV) of around 8.6%. However, the DNA histograms of cells stained with propidium iodide had a higher CV value, a poor fit degree, and no obvious G1 and G2/M peaks. The results indicate that SYBR Green I is an adequate detection method for nuclear DNA quantitation in cells of P. tricornutum using a flow cytometer equipped with a 488-nm laser, and without RNase A treatment.

关 键 词：三角褐指藻 流式细胞术 碘化丙啶(PI) SYBR-green I 细胞核DNA染色 

分 类 号：Q179.1[生物学—水生生物学] Q2-33[生物学—普通生物学]