流式细胞仪测定荔枝倍性和基因组大小的细胞核提取液筛选与应用  

Nuclear isolation buffer optimization and the determination of ploidy level and genome size of litchi using flow cytometry

作　　者：赖彪 吴传龙[2] 秦永华[2] 刘成明[2] 冯奇瑞[2] 叶自行[2] 胡桂兵[2] 王惠聪 LAI Biao;WU Chuanlong;QIN Yonghua;LIU Chengming;FENG Qirui;YE Zixing;HU Guibing;WANG Huicong(School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China;College of Horticulture,South China Agricultural University, Guangzhou 510642, Guangdong, China)

机构地区：[1]长江师范学院现代农业与生物工程学院,重庆涪陵408100 [2]华南农业大学园艺学院,广州510642

出　　处：《果树学报》2019年第7期939-946,共8页Journal of Fruit Science

基　　金：国家现代农业产业技术体系建设专项资金资助项目“国家荔枝龙眼产业技术体系”(CARS-33);长江师范学院校级科研项目(2016KYQD20和2016XJQN06)

摘　　要：【目的】流式细胞术是目前测定植物倍性和基因组大小差异的最快、最有效的方法。建立适合荔枝的流式细胞术的方法,对荔枝倍性育种以及基因组大小的确定是必不可少的。【方法】笔者以荔枝的幼嫩叶片为材料,筛选适合荔枝的细胞核提取液配方,建立利用流式细胞仪测定荔枝倍性和基因组大小的方法。用改进的标准两步法,比较了6 种常用细胞核提取液提取细胞核的效果。【结果】利用WPB(Woody Plant Buffer)提取的大部分荔枝品种(品系)叶片细胞核稳定、分辨率高、细胞碎片少且细胞G0/G1峰的变异系数(CV)较低,平均CV值为5.12%,说明该配方适用于酚类物质丰富的荔枝细胞核的提取。同时以已知染色体数量的‘无核荔’(2n=30)为外标,检测了18 个品种(品系)的倍性,发现参测样品的G0/G1峰与‘无核荔’G0/G1峰的荧光均值的比值为0.78~1.24,说明所测荔枝品种(品系)均为二倍体。以已知基因组大小的‘Stupické polní rané’番茄为内标测定了14 个品种(品系)的基因组大小,结果表明荔枝基因大小约为550~620 Mb,平均602 Mb,不同荔枝品种(品系)间基因组大小存在一定差异。【结论】WPB细胞核提取液提取的荔枝幼嫩的叶片的细胞核质量好,可用于流式细胞术荔枝倍性和基因组大小的测定,测定的结果显示参测荔枝品种(品系)均为二倍体,无单倍或多倍的情况,不同荔枝品种基因组大小有一定的差异。【Objective】Flow cytometry is particularly applicable in the determination of ploidy and genome size of diverse plant samples because it is convenient, fast and reliable. The sample preparation in flow-cytometry determination is relatively simple and less costly. The nuclei can be counted in a short time using a sample volume of only a few milligrams of plant tissues. Although chromosome counting has been used in ploidy determination in plants, in many cases representative picture of a cell population is difficult to obtain and the determination is inefficient. DNA flow cytometry requires preparation of the suspensions of intact nuclei, and suitable nuclei isolation buffer is a bottleneck for litchi ploidy and genome size estimation. The main problem is that it is not easy to prepare suspensions of intact nuclei from the leaves of woody plants, which contain cells with a rigid cell wall and secondary metabolites. This study aimed to choose the best nuclei isolation buffer and establish an appropriate flow cytometry method for litchi ploidy and genome size estimation.【Methods】In this study, young litchi leaves were selected as material to compare the nuclei isolation efficiency and extraction quality among six frequently-used buffers, including Otto’s, MgSO4, Tris-MgCl2, Galbraith’s, WPB and GPB buffer, using modified two step procedures. First, place a small amount of young litchi leaf (typically 50-100 mg) in the center of a glass petri dish, which was placed on ice to keep the sample cold. Then add 2 mL ice-cold nuclei isolation buffer to the petri dish and chop the tissue immediately in the buffer with a razor blade (use new one for each sample). Filter the chopped solution through a 53 mm nylon mesh into a new a 2 mL Eppendorf tube, which was centrifuged (1 000 r·min^-1) for 5 minutes at 4 ℃ and the supernatant was carefully remove. The nuclei in the sediment was re-suspended by gentle shaking in 400 μL new nuclei isolation buffer supplemented with 50 μg·mL^-1 PI (Propidium iodide) and 50 μg

关 键 词：荔枝 流式细胞术 细胞核提取液 倍性 基因组大小 

分 类 号：S667.1[农业科学—果树学]