利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

Chromosome Painting in Secale cereale with PCR Products from Microdissected Chromosome 1R

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后，再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针，与其根尖细胞中期分裂相进行染色体原位杂交，发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列，而其信息量却较黑麦总基因组少；当以适量的黑麦基因组ＤＮＡ进行封阻时，微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上，说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外，以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针，染色体原位杂交检测发现，这一高度重复序列可能为端粒相关序列；而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系，为染色体微克隆技术在植物中的进一步应用提供了便利。

Microdissected 1R chromosomes of rye ( Secale cereale L.) were amplified by Sau 3A linker adaptor mediated PCR (LA_PCR) for two rounds. After demonstration by Southern hybridization, that their PCR products were originated from the genome of rye, the second round PCR products of the chromosome 1R, rye genomic DNA and rDNA were used as probes to hybridize in situ with metaphase chromosomes of root_tip cells. It was found that the PCR products from microdissected 1R chromosomes included a large amount of 1R chromosome_nonspecific repetitive sequences. However, its information capacity was fewer than the total rye genome. While blocking with proper amount of genomic DNA, the second round PCR products from the microdissected 1R chromosomes were successfully relocated to the pair of 1R chromosomes in mitotic metaphase, indicating that the PCR products certainly contained 1R chromosome specific fragments. In addition, a highly repetitive sequence and a low/single copy sequence selected from the microclone library of chromosome 1R were used as probes. Chromosome in situ hybridization inspected that the repetitive sequence probably was a telomeric relative sequence. But no signal was detected with the low/single copy probe. These data suggest that chromosome painting is a useful way to confirm the origination of microdissected chromosomes and to screen chromosome_specific probes. This research has established a consulted experimental system for plant chromosome painting, which could provide convenience for further applications of chromosome microcloning techniques in plants.