莲藕根尖染色体制片与免疫荧光染色技术的优化  

The Optimization of Chromosome Preparation and Immunofluorescence Staining for Root Tip of Nelumbo nucifera

作　　者：王海燕[1,2] 朱之轩 金晶[2] 丁毅[2] 

机构地区：[1]新疆教育学院科学教育学院,乌鲁木齐830043 [2]武汉大学生命科学学院遗传学系,武汉430072

出　　处：《生物技术通报》2016年第4期74-79,共6页Biotechnology Bulletin

基　　金：国家自然科学基金资助项目(31271310)

摘　　要：在细胞遗传学研究中,莲藕根尖染色体制片比较困难,研究了莲藕根尖染色体制片过程中适宜取材的根尖长度、有丝分裂指数、预处理试剂和处理时间、固定时间、酶解与低渗时间等相关因素,并对莲制片进行了免疫荧光染色。结果表明:根尖长为1.5 cm左右时有丝分裂指数最高,预处理采用冰水冷冻处理24 h分裂相较多,染色体形态清晰;用4%多聚甲醛固定1h,染色体形态较好;采用2.5%纤维素酶和2.5%果胶酶混合液37℃消化30 min,酶解较彻底,细胞质残留少,染色体分散良好;1×PBS低渗30 min,染色体形态较好;免疫荧光染色后经检测可观察到较强的信号。It is difficult to prepare lotus＇ s（Nelumbo nucifera）root tip chromosome in the cytogenetic study. Therefore,some related factors were studied,such as selecting the proper length of root tip,mitotic index,pretreatment medicament and time,the fixing time,the time of enzyme hydrolysis,and low osmosis in the chromosome preparation from the root tip cells of lotus,and then the immunofluorescence staining was also carried out to validate the chromosome preparation. The results showed that the mitotic index was the highest and morphology was clear when the root tips length was approximately 1.5 cm and the root tips was treated with ice water for 24 hours. The chromosome morphology was solid while root tips was for 1 h fixing with 4% paraformaldehyde. The enzyme hydrolysis was complete,the residue of cytoplasm was little,and the chromosome scattered well while the tips were digesting 30 min with combination of 2.5% cellulase and 2.5% pectinase at 37℃. The chromosome morphology was fine under 30 min low osmosis treatment by 1×PBS. Strong signals were detected after the immunofluorescence staining. This study set up the foundation for the relevant researches on cytogenetic and epigenetic studies of N. nucifera.

关 键 词：莲藕根尖细胞 预处理 有丝分裂指数 染色体制片 DAPI染色 免疫荧光 

分 类 号：S645.1[农业科学—蔬菜学]