水仙单染色体特异文库的构建  

Construction of Specific Library of Narcissus Monosomes

作　　者：吴菁华[1] 吕柳新[1] 

机构地区：[1]福建农林大学园艺学院,福州350002

出　　处：《西北植物学报》2006年第2期305-308,共4页Acta Botanica Boreali-Occidentalia Sinica

基　　金：福建农林大学青年教师基金项目(05B02)

摘　　要：采用玻璃针分离法，通过显微操作系统成功地分离到一个多花水仙（Narcissus tazetta L.2n=22）品种的中着丝粒单染色体，将分离到的单染色体放入0．2ml。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0．3～3．0kb之间的DNA片段。用水仙基因组DNA标记作探针。与扩增产物进行Southern杂交，从而证明单染色体DNA确实已被成功地扩增。将第二轮PCR产物构建质粒文库，随机挑取90个重组子进行分析。发现插入片段主要在600～2500bp之间。Narcissus monosomes with middle centromeres were isolated with glass needles with the help of micromanipulator and then put into 0. 2 mL Eppendorf tubes;next ,the monosomes were deproteinised and digested with Sau3A;afterwards,the DNA fragments of the monosomes were ligated with Sau3A linkers at their two ends and PCR amplified in two rounds; after the amplification, DNA fragments of 300 -3 000 bp was obtained. With genomic DNA maker as the probe, the southern blotting of the amplification products was conducted, which proved that monosome DNA were indeed successfully amplified. The products of the second round amplification were micro-cloned to construct the plasmid library and the analysis of 90 randomly chosen clones found that the inserted fragment ranged between 600-2 500 bp.

关 键 词：水仙 微分离 单染色体 微克隆 

分 类 号：Q785[生物学—分子生物学] Q247