甘蓝染色体及伸长DNA纤维制备技术的研究
An Efficient Method for Preparation of Chromosomes and Extended DNA Fibers in Brassica oleracea
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摘要: 以甘蓝根尖、花药、黄化苗等为材料,初步建立了甘蓝有丝分裂前中期染色体、减数分裂粗线期染色体以及伸长DNA纤维等3种具有不同空间分辨率的靶DNA载体的制备技术.结果表明:通过变温处理可以有效提高前中期染色体分裂相比率;采用0.002 mol/L 8-羟基喹啉20℃预处理1 h、2，纤维素酶和2，果胶酶混合液37℃酶解约2 h、0.075 mol/L氯化钾25℃低渗30 min,制备的前中期染色体效果最佳;2，纤维素酶和2，果胶酶混合液37℃酶解约3 h,可获得伸展良好、背景干净的粗线期染色体;分子梳理法与移动界面法相比,前者制备的伸长DNA纤维效果较好.此方法的建立为荧光原位杂交技术在甘蓝相关研究领域的应用打下了坚实的基础.
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关键词:
- 甘蓝,染色体,伸长DNA纤维,制片
Abstract: A method of preparation was established for different FISH (fluorescence in situ hybridization)resolution on prometaphase and pachytene chromosomes and extended DNA fibers with roots, anthers and albino seedlings of Brassica oleracea. Alternating temperature treatment effectively improved the divisionindex of prometaphase cells. Optimum results were obtained for the preparation of prometaphase chromosomes when pretreatment was made with 0. 002 mol/L 8-hydroxy quinoline at 20 ℃ for 1 h before treatment with the enzyme mixture of 2% cellulase and 2% pectase at 37 ℃ for approximately 2 h in combination with a hypotonic treatment with 0. 075 mol/L potassium chloride at 25 ℃ for 30 min. Well-spread pachytene chromosomes with clear background were obtained through the treatment with the enzyme mixture of 2% cellulase and 2% pectase at 37 ℃ for about 3 h. Molecular combing was better than moving interface for preparing extended DNA fibers. The method lays a solid foundation for the application of FISH in B. oleracea. -
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