大白菜适于FISH的染色体制片技术研究  

Studies on the Chromosome Preparation for FISH in Chinese Cabbage

作　　者：李玉玺[1] 轩淑欣[1] 王彦华[1] 李晓峰[1] 李九欢[1] 申书兴[1] 

机构地区：[1]河北农业大学园艺学院,河北保定071001

出　　处：《中国农学通报》2011年第10期284-288,共5页Chinese Agricultural Science Bulletin

基　　金：国家教育部博士点基金"大白菜BAC文库的构建及其在初级三体的BAC-FISH定位"(20101302110001);河北省青年科学基金"大白菜染色体特异BAC-FISH标记的开发和候选基因的物理作图"(C2010000738)

摘　　要：采用酶解去壁低渗法进行大白菜染色体制片,研究了影响其有丝分裂相指数和制片质量的根尖长度、预处理药剂和时间、前低渗和酶解的温度与时间等相关因子,并对大白菜制片质量进行了25SrDNA的FISH检测。结果表明:当大白菜生长旺盛的根尖长为1.5～2.0cm时中期分裂相指数最高;最佳预处理药剂为0.002mol/L8-羟基喹啉,当预处理时间为2h时能获得较多的分裂相和清晰的染色体形态;0.075mol/LKCl中25℃前低渗0.5h,然后采用2%纤维素酶和2%果胶酶混合液4℃消化5～6h或25℃消化2～2.5h,染色体分散良好,酶解完全,无细胞质残留,制备的染色体标本可不经预处理直接用于FISH杂交。本研究结果为在大白菜上开展FISH技术的相关研究奠定了基础。Adopting the wall degested with enzyme and low osmotic method to prepare the chromosome production of Chinese cabbage,some related factors effecting on the mitotic index and chromosome preparation quality were studied,including the root tip length,pretreatment medicament and time,the tempreture and time of pre-low osmotic and enzyme hydrolysis,and fluorescencein situ hybridization was also used to detect the chromosome preparation quality using the 25S rDNA as probe.The results showed that the best method was treating the root tips with 0.002 mol/L 8-hydroxyquinoline for 2 h when the root length was 1.5-2 cm,0.075 mol/L KCl at 25℃ for 0.5 h pre-low osmosis treatment,and the optimum conditions for enzyme hydrolysis were at 4℃ for 5-6 h or 25℃ for 2-2.5 h with 2% cellulase and 2% pectinase.The high quality chromosome preparation with scattering chromosomes and clean background was achieved by above method,and could be used to FISH without needing pretreatment.This study will set up the foundation for the related research about FISH in Chinese cabbage.

关 键 词：大白菜 分裂相指数 染色体制片 FISH 

分 类 号：S634.1[农业科学—蔬菜学]