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。
In this research, in order to establish a FCM system in Quercus, four common nuclear isolation buffers ( Tris- MgCI2, 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-MgC12 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 retool/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 6n field population of Quercus by FCM.