向日葵单染色体显微分离及特异文库的构建.pdfVIP

中国细胞生物学学报 Chinese Journal of Cell Biology 2014, 36(1): 33–38 DOI: 10.11844/cj cb.2014.01.02 10 向日葵单染色体显微分离及特异文库的构建 1 1,2 1* 孙瑞芬 闫素丽 安玉麟 1 2 ( 内蒙古农牧业科学院生物技术研究中心, 呼和浩特010031; 内蒙古农业大学农学院, 呼和浩特010019) 摘要 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~ 1 500 bp 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 5 5 为2.26×10 和2.57×10 。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200~700 bp和 200~500 bp, 平均插入片段大小分别为535 bp和480 bp 。这是染色体微分离与微克隆技术首次在向 日葵上的应用。 关键词 向日葵; 染色体; 显微分离; PCR扩增; 单染色体文库 Microdissection of Single Chromosome and Construction of Its DNA Library in Sunflower (Helianthus annuus L.) 1 1,2 1* Sun Ruifen , Yan Suli , An Yulin 1 ( Biotechnology Research Center, Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences, Huhhot 010031, China; 2Institute of Agronomy , Inner Mongolia Agriculture University , Huhhot 010019, China) Abstract 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 microclo