向日葵单染色体显微分离及特异文库的构建

Microdissection of Single Chromosome and Construction of Its DNA Library in Sunfl ower(Helianthus annuus L.)

采用玻璃针分离法，通过显微操作系统成功地分离到内葵杂3号三交种和单交种的随体染色体，经两轮LA．PCR扩增得到250～1500bp的DNA片段。用各自的基因组DNA标记成探针，与随体染色体扩增产物进行Southern杂交，显示杂交信号，证明内葵杂3号三交种和单交种随体染色体DNA已被成功扩增。将第2轮PCR产物构建质粒文库，得到三交种和单交种克隆数分别约为2．26×10^5和2．57×10^5。各随机挑取30个重组子进行分析，发现插入片段大小分别为200-700bp和200～500bp，平均插入片段大小分别为535bp和480bp。这是染色体微分离与微克隆技术首次在向日葵上的应用。

The satellite chromosome of triple cross and single cross of ＂Inner Mongolia Hibrid oil sunflower 3＂ were isolated by glass needle method with the help of micromanipulator. After two rounds of LA-PCR amplification, the PCR products ranging from 250 to 1 500 bp were obtained. Southern blot with genomic DNA revealed hybridization signal, indicating that DNAs from the satellite chromosome had been successfully amplified. The second-round PCR products were microcloned into plasmid vector, and their DNA libraries were constructed. About 2.26×10^5 and 2.57×10^5 recombinant clones were obtained from triple cross and single cross of sunflower respectively. 30 clones were randomly selected for size evaluation. The results demonstrated that the size of the DNA fragmenrts from triple hybrid sunflower varied approximately from 200 to 700 bp with an average of 535 bp, and the DNA fragmenrts from single cross hybrid sunflower varied approximately from 200 to 500 bp with an average of 480 bp. The microdissection and microcloning technoloev has been aonlied forsunflower chromosome analysis.