银杏第1染色体DNA文库的构建

Construction of the first chromosome DNA library of ginkgo

观察了50个银杏根尖的染色体,发现在约半数根尖中,最大的1对染色体(第1染色体)随体有差异,其中1条随体较大而明显,另1条则很小.采用玻璃针分离法,通过显微操作系统成功地分离了随体较为明显的那条第1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2轮PCR扩增,得到了大小为300-3000bp的扩增片段.用第2轮PCR产物构建质粒文库,得到了约含有75000个重组子的该染色体DNA文库.随机挑取66个重组子进行分析,发现插入片段大小主要分布在500-2000bp,平均为800bp.该文库为银杏第1染色体特异探针的筛选、遗传图谱的构建、重要基因的克隆以及性染色体的鉴别等研究提供了基础.

Fifty root tips of ginkgo (Ginkgo biboba var. huana) were observed. It was found that in about half of the roots, the satellites of the two first chromosomes were different in size, one was larger and the other was smaller. The first chromosome with bigger satellite was isolated by micromanipulation with glass needles and digested with Sau3A. Both ends of DNA fragments were ligated with Sau3A linker adopters. After two rounds of PCR amplification, DNA fragments ranging from 300-3 000 bp were acquired. The secondround PCR products were cloned into plasmid vectors to generate a chromosomespecific library consisting of approximately 75 000 recombinant clones, from which 66 clones were randomly selected for size evaluation. The results showed that the size of the inserts varied from 500-2 000 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.