甘蓝2号染色体的高分辨率5S rDNA荧光原位杂交

 【目的】羽衣甘蓝5S rDNA 的染色体定位和拷贝数分析,为进一步利用FISH进行2号染色体基因定位和细胞遗传图谱构建奠定基础。【方法】以羽衣甘蓝为材料,采用荧光原位杂交技术将DIG标记的5S rDNA探针定位于不同分辨率的绒毡层细胞中期染色体、粗线期染色体以及伸长DNA纤维上。【结果】在中期染色体和粗线期染色体上,都同时获得3个杂交信号位点（a、b、c）,且位于2号染色体的长臂近着丝粒区域,其信号强度为b＞a＞c;而在伸长DNA纤维上,出现了3种不同长度的念珠状长链（a、b、c）, 其物理大小分别为257、359和134 kb,这3种长链分别与3个信号位点形成一一对应关系。【结论】在羽衣甘蓝2号染色体上存在3个串联重复位点,粗略估算出3个5S rDNA位点的拷贝数分别为510、712和266。

Study on High-Resolution 5S rDNA-FISH of Brassica oleracea Chromosome2

Objective] The aim of this experiment is to localize 5S rDNA on the chromosomes of Brassica oleracea var.acephala by FISH(fluorescence in situ hybridization)and estimate the copy number of 5S rDNA,further provide a basis for the location of genes and the construction of cytogenetic map of chromosome 2 by FISH.Method]B.oleracea Val.acephala was used as the experimental material,5S rDNA probe was labeled by PCR-DIG(PCR-digoxigenin).By FISH,5S rDNA probe was located on the metaphase chromosomes of tapetal cell,pachytene chromosomes and extended DNA fibers(EDF).Result]In the metaphase chromosomes of tapetal cell and pachytene chromosomes,it Was observed that three closely adjacent 5S rDNA hybridization signal sites(a,b,C)located near the centromere in the long arm of the submetacentric chromosome 2,the intensity of signals is b＞a＞c.In the extended DNA fibers,it Was estimated that the physical size of three different stretches of beads-on-string(a,b,C)is of the order of 257 kb,359 kb,and 134 kb,respectively.Conclusion]It was proved that B.oleracea var.acephala contains three tandem repeat sites,and estimated that the copies number of three 5S rDNA locus ale about 510,712,and 266,respectively.