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植物细胞壁及细胞质的存在和植物染色体所具有的高浓缩特性,限制高效率原位杂交定位在植物细胞内的进行。针对小型染色体芸薹属植物采用常规方法DNA制备纤维的效果不佳的特点,新建制备方法:利用减数分裂前期的染色体为材料, 在硅化的玻片上先后通过蛋白酶解和乙醇:乙酸(3:1)的适当处理,采用移动界面法制备EDFs。制备的EDFs比未经伸长处理的染色体在经向和横向方面分别取得较高程度的伸长与膨胀,长度可达到89-257 gm,比相应地中期染色体增长30-107倍, 分辨率可达42．8-53．0 kb。利用SRK和SCR两种探针同时在甘蓝粗线期染色体和EDFs上进行了原位杂交,首次鉴定了S基因座在其单倍体基因组中单拷贝性。在杂交信号检测中尽管未经过信号放大,但仍然可以观察到清晰的绿色信号;经荧光显微镜观察,在单一的EDF上发现两个相距1 μm的SCR和SRK的信号点,由此得出局部分辨率为4 kb的最高伸长度。
The presence of plant cell walls and cytoplasm and the high concentration of plant chromosomes limit the efficient in situ hybridization localization within plant cells. Aiming at the characteristic that the small chromosome Brassica plant adopts the conventional method to prepare the fiber with poor effect, the preparation method of the new method uses the chromosome at the pre-meiotic stage as the material, sequentially passes through proteolysis and ethanol: acetic acid (3: 1) the appropriate treatment, the use of mobile interface preparation of EDFs. The prepared EDFs obtained a higher degree of elongation and expansion respectively in the warp and transverse directions than the non-elongated chromosomes, reaching lengths of 89-257 gm, which was 30-107 times longer than that of the corresponding metaphase chromosomes. The resolution was Up to 42.8-53.0 kb. SRK and SCR probes were used to perform in situ hybridization on pachytene chromosomes and EDFs. It was the first time that the S locus was identified as a single copy in the haploid genome. In the detection of hybridization signal, although no signal amplification, but still can be observed clear green signal; observed by fluorescence microscopy, found in a single EDF and the SRK SRK 2 km away from the signal points, which come to the local Maximum resolution of 4 kb.