西瓜第一染色体显微分离及其特异文库的构建

采用玻璃针分离法,通过显微操作系统成功地分离到西瓜(2n=22)的第一单染色体,将分离到的西瓜染色体放入0.2 mL Eppendorf管中,经去蛋白,Sau3A酶切,并在染色体DNA片段两端加上Sau3A人工接头后,进行两轮PCR扩增,得到0.4～2.0 kb之间的DNA片段.用西瓜的基因组标记成探针,与单染色体扩增产物进行southem杂交,表明这些西瓜单染色体扩增片段与西瓜基因组DNA之间有同源性,从而证明单染色体DNA确实已被成功地扩增.将第2轮PCR产物构建质粒文库,得到约20 000个重组子.这是染色体微分离、微克隆技术首次在瓜类作物中的应用,该方法为直接从西瓜单条染色体DNA文库中筛选分子标记奠定了基础,说明利用染色体显微分离、克隆技术对西瓜等瓜类作物基因组进行研究是可行的.

Microdissection of the First Chromosome and Construction of Its DNA Library in Watermelon

The entire chromosome 1 was microdissected by using a fine glass needle under the inverted microscope, and then transferred into a collection chamber. Procedure for chromosome preparation was the squash method, in which the root-tip cells of watermelon cultivar "Zhongyuyihao" were separated and spread. The microdissected chromosome adhered to the needles was digested with proteinase K, and its DNA was extracted and digested with Sau3A. DNA ligation to a linker/primer set and PCR was carried out, in which the sequence of the linker was 5'-GATCCTGAGCTCGAATTCGACCC-3' and 5'-GGGTCGAATTCGAATTCG AGCTCAG-3]. After two rounds of PCR amplification, a smear of DNA fragments ranging from 0.4 to 2.0 kb were generated. Southern hybridization result showed that the PCR products from the single watermelon chromosome was homogeneous with the watermelon genomie DNA, indicating that DNAs from the chromosome have been successfully amplified. The second-round PCR products from chromosome of watermelon were microcloned to construct the plasmid library, including 20 000 recombinant clones. It is first reported that a simple procedure for preparation and microdissection of small chromosomes in watermelon has been successfully established. In addition, this study demonstrates that chromosome microdisseetion and cloning can be applied in genomic study of melon crops.