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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.
The preparation technology of three kinds of target DNA vectors with different spatial resolution was preliminarily established using the apical kale, the anther and the yellowing seedlings as materials, and preliminary establishment of the pre-mitosis mitochondrial metamerism, the meiosis of the mitochondrial pachytene and the elongation DNA fiber The results showed that the ratio of chromosome cleavage phase in the early and middle stages could be effectively increased by temperature-changing treatment. The enzymatic activity of 37 ℃ enzyme of 2% cellulase and 2% pectinase mixture was pretreated with 0.002 mol / L 8-hydroxyquinoline at 20 ℃ for 1 h. About 2 h, 0.075 mol / L potassium chloride at 25 ℃ for 30 min, the best pre-metaphase chromosome was obtained. When the mixture of 2% cellulase and 2% pectinase was hydrolyzed at 37 ℃ for 3 h, Good and clean background of pachytene chromosomes.Compared with the mobile interface method, molecular combing method showed better elongation DNA fiber effect.The establishment of this method has laid a solid foundation for the application of fluorescence in situ hybridization in the field of cabbage related research Foundation.