【摘要】 以欧洲山杨(Populustremula)根尖细胞为材料,采用玻璃针分离法,通过显微操作器成功地分离出一号染色体。将分离到的单条染色体去蛋白、Sau3A酶切,并在染色体DNA片段两端加上Sau3A人工接头,进行两轮PCR扩增,得到了200~3000bp的DNA扩增片段。以DIG-dUTP标记的欧洲山杨基因组DNA为探针进行Southern杂交,结果表明显微分离出的染色体扩增片段与欧洲山杨基因组DNA同源,从而证明一号染色体DNA确实已被成功地扩增。以一号染色体第2轮PCR产物为探针进行荧光原位杂交,发现荧光信号较密集的分布于一号染色体,但同时荧光信号也出现在其它染色体的着丝粒及端粒区域。对第2轮PCR产物进行克隆,构建单条染色体DNA文库,经分析,该微克隆文库包含约3×105个重组子。随机挑选160个重组子进行鉴定,证明该文库的插入片段主要介于230~2200bp之间,平均800bp。该文库的构建为欧洲山杨1号染色体特异探针的筛选、遗传图谱的构建、重要基因的克隆等研究奠定了基础。更多还原

【Abstract】 This study was performed to establish a method for single chromosome microdissection and microcloning in forest plants using poplar(Populus tremula) as a model. An individual chromosome 1 was microdissected from the metaphase spreads of poplar root-tip cells with the fine glass needle controlled by a micromanipulator. The dissected chromosome was amplified in vitro by the Sau3A linker adaptor mediated PCR technique, by which 200 bp to 3 000 bp smear DNA fragments were obtained. Southern hybridization result showed the PCR products from the single poplar chromosome were homogeneous with the poplar genomic DNA, indicating that DNA from the single chromosome has been successfully amplified. Then, he second round PCR products were used as a complex probe mixture for fluorescent in situ hybridization (FISH) on the metaphase spreads of poplar. Hybridization signals were observed, mainly, along the entire chromosome 1, at the same time, signals were also present on telomeric and centromeric regions of other chromosomes. The second round PCR products from the single chromosome 1 were cloned into T-easy vectors to generate a DNA library of the chromosome 1. Approximately 3×10~5 recombinant clones were obtained. Evaluation based on 160 randomly selected clones showed that the sizes of the cloned inserts varied from 230 bp to 2 200 bp with an average of 800 bp. This library will facilitate specific probe screening, molecular map construction, gene tagging and gene cloning on this chromosome.更多还原