水稻中期染色体和DNA纤维的高效制备技术

The Highly Efficient Methods for Preparation of Rice Metaphase Chromosomes and DNA Fibres

水稻中期染色体和DNA纤维制备是水稻分子细胞遗传学研究中的关键技术。目前,这两个技术还有很多不足,该研究建立了高效制备水稻中期染色体和DNA纤维的方法。该方法制备的染色体,分裂相多、杂质少、背景清晰、染色体分散且形态好,水稻根尖分生组织细胞的分裂指数高达25%。植物细胞的细胞壁是制备DNA纤维的最大障碍,所以必须先提取细胞核,然后裂解细胞核释放出DNA纤维。在这个研究中,还建立了一个用刀切法分离细胞核,进而用SDS裂解核膜,用载玻片拖出DNA来制备水稻DNA纤维的方法。该方法制备的DNA纤维多呈平行的细线,背景清晰,伸展的程度均匀,适合于原位杂交。

The preparation of mataphase chromosomes and DNA fibres of rice is the key technique in the research of rice genome structure. However these techniques are still in deficiency at present. In this study, a method to prepare metaphase chromosomes with high efficiency was deve-lopped in rice. In the chromosome slides prepared using this method, there were mass of metaphase cells, less impurity, clear background, well dispersed chromosomes and well presered chromosome morphologies. The mitotic index of root cells is up to 25%. We also established a DNA fibres preparing method. Since cell wall is a main abstacle in the preparation of DNA fibres, thus nuclei ware isolated by chopping first, then lysed by SDS to release DNA, and DNA fibers were prepared by drawing DNA using a cover side. DNA fibres prepared by this method showed equally spread parallel thread with clear background, and were suitable for in situ hybridization analysis. The highly efficient methods to prepare rice metaphase chromosomes and DNA fibres will surely accelerate rice genome mapping and organization analysis.