摘要
采用碱性低渗结合高氯仿一步显带技术获得了斑马鱼二价体高分辨多重G带 ,所出现的带纹丰富 ,反差明显。采用地高辛为标记的以限制性内切酶AluⅠ介导的原位切口平移技术使斑马鱼二价体上呈现出类G带的多重带 ,获得了明暗反差强烈的带纹结果。通过比较多个不同分裂相的多条染色体 ,发现带纹的分布具明显特征性 ,并且特征一致 ,带纹数目基本吻合。首次从方法学上对斑马鱼二价体的制备过程及多重带显带技术进行了分析、探讨和总结 ,力求将该技术程序化、系统化 ,使其具有可重复性和可操作性 ,并对显带的可能机理进行了讨论。
The pachytene bivalents with high-resolution multiple G bands of zebrafish were obtained after the treatment with alkaline hypotonic solution and high concentration of chloroform fixative solution.When comparing six group chromosomes from different pachytene specimens,the characteristic and the number of bands were well matched.In order to systematize this technique and get stable result,we summarize the preparation procedure of the zebrafish bivalents.The 6-month-old to one-year-old zebrafish whose spermary appears ivory-white and opaque,is good material.The whole testis should be treated with hypotonic solution for 1.5～2 h at room temperature.Then,the testes were fixed for 20 min in high chloroform fixative solution (chloroform∶[KG-2]methanol∶[KG-2]acetic acid,3∶[KG-2]6∶[KG-2]1),and fixed in Carnoy’s solution (methanol∶[KG-2]acetic acid,3∶[KG-2]1) for two times.In addition,with the treatment of restriction endonuclease AluⅠ directed in situ nick translation,we successfully obtained well-resolved restrictive endonuclease banding of zebrafish bivalents,which was considered as G-like band patterns.The aging of the specimen is also important factor,should let them dry at room temperature for one week.The application of these methods in cytogenetics research of zebrafish and other fish can be expected.Construction of the steady technique system to prepare high resolution banding bivalents and idiogram of zebrafish is the basement to found stable and accurate framework for physical map.
基金
国家自然科学基金 (批准号 :3 0 170 5 0 8)
国家自然科学基金委主任基金 (批准号 :3 0 15 0 0 5 )资助项目~~
