为建立适于栎属植物的流式细胞核实验体系,分别利用Tris-MgCl2、Galbraith’s、Marie’s和Otto’s细胞核分离缓冲液对麻栎、栓皮栎、白栎和短柄枹栎制备细胞核悬浮液。检验发现:Tris-MgCl2、Galbraith’s缓冲液所制备样品未能检测出DNA峰,Otto’s缓冲液制备的样品中,除白栎外其他3个样品的测试结果均较差,Marie’s缓冲液所制备的4个样品测试DNA峰的变异系数(CV)均大于10%。采用正交设计方法对经典Marie’s缓冲液中的β-巯基乙醇和Tween 20组分进行了优化,发现麻栎、栓皮栎、白栎和短柄枹栎的最适β-巯基乙醇浓度和Tween 20体积比分别为45 mmol/L和2.5%、15 mmol/L和2.0%、60 mmol/L和1.0%及30 mmol/L和1.0%。运用改进后的最适Marie’s缓冲液进行流式细胞核测定,4种样品的变异系数均显著下降(在3.52%～4.81%之间),表明改进后的Marie’s缓冲液对4种栎属植物的测试结果准确可信。
Abstract
In this research, in order to establish a FCM system in Quercus, four common nuclear isolation buffers(Tris-MgCl2, Galbraith’s, Marie’s and Otto’s)were used to prepare samples from mature leaf tissues of four wild oaks(Quercus acutissim, Q. variabilis, Q. fabri, Q. glandulifera var. brevipetiolata). Then all samples were tested on flow cytometer. No DNA peak was detected in the samples treated with Tris-MgCl2 and Galbraith’s buffers. Samples prepared with Otto’s buffer, except for Q. fabri, showed poor results. The coefficients of variation(CV)of the DNA peak of the 4 samples prepared with Marie’s buffer were larger than 10%. An orthogonal design was used to optimize conventional Marie’s buffer in two factors: β-mercaptoethanol and Tween 20. Results indicated that the most suitable concentration of β-mercaptoethanol and Tween 20 on Quercus acutissim, Q. variabilis, Q. fabri, Q. glandulifera var. brevipetiolata respectively was 45 mmol/L,2.5%; 15 mmol/L, 2.0%; 60 mmol/L, 1.0%; 30 mmol/L, 1.0%. CVs for Quercus DNA peak found had decreased obviously to 3.52%-4.81% by applying modified Marie’s buffers. It proved that the test results of the four Quercus samples prepared with modified Marie’s buffers were accurate and dependable. This research laid a foundation for a wide range of studies on field population of Quercus by FCM.
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基金
收稿日期:2013-12-19 修回日期:2014-08-19
基金项目:国家自然科学基金项目(31370666); 江苏高校协同创新计划; 江苏高校优势学科建设工程资助项目(PAPD)
第一作者:魏高明,博士生。*通信作者:方炎明,教授。E-mail: jwu4@njfu.edu.cn。
引文格式:魏高明,方炎明. 栎属植物流式细胞术实验体系的建立与优化[J]. 南京林业大学学报:自然科学版,2015,38(1):167-172.
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