水稻BAC在玉米有丝分裂染色体上FISH杂交体系的构建

Constructing FISH System with Rice BAC Probes on Mitotic Chromosomes of Maize

以水稻细菌人工染色体(BAC)为探针在玉米有丝分裂的细胞学制片上进行荧光原位杂交(FISH),探讨玉米基因组CotDNA对BAC探针重复序列的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化、水稻BAC探针的特异性重复序列的封阻对FISH杂交信号特异性的影响.初步形成了一套以水稻BAC探针在玉米有丝分裂染色体上进行BAC-FISH杂交的优化技术体系.研究结果表明使用玉米基因组CotDNA来封阻水稻BAC探针的重复序列玉米基因组CotDNA的Cot值应小于50,同时还需根据不同探针调整CotDNA的Cot值及与探针的比例;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻BAC探针本身特异的重复序列的封阻对BAC-FISH杂交信号特异性的改善具有较好的效果.

Fluorescence in situ hybridization （FISH） using the rice BAC-DNA as a probe had been performed on mitotic chromosomes. To optimize the BAC-FISH technique, the following parameters were investigated, they were signal specificities of FISH by the maize genomic C0 t DNA blocking effects on repetitive sequences of the BAC-DNA, washing temperature, FAD concentration in washing buffers and hybridization mixtures, and blocking specific repetitive sequence of the rice BAC. The results showed that C0t DNA of maize genomic blocked the repetitive sequence of the rice BAC-DNA when the C0 t value was below 50. Meanwhile, it was necessary to adjust C0 t value according to the different probes and their ratios. Decreasing the concentration of FAD in hybridization mixtures, adjusting the washing rate after hybridization, especially blocking the rice specific repetitive sequences of BAC-DNA, could improve the positive signals of BAC-FISH.