栎属植物流式细胞术实验体系的建立与优化  

Establishment and optimization of an experiment system for flow cytometry in Quercus

作　　者：魏高明[1] 方炎明[1] 

机构地区：[1]南京林业大学生物与环境学院,江苏南京210037

出　　处：《南京林业大学学报（自然科学版）》2015年第1期167-172,共6页Journal of Nanjing Forestry University：Natural Sciences Edition

基　　金：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)

摘　　要：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.

关 键 词：栎属植物 成熟叶片 流式细胞术 细胞核分离缓冲液 

分 类 号：S792.18[农业科学—林木遗传育种]