白氏文昌鱼单个体基因组BAC文库的构建

Construction of genome BAC library for single Branchiostoma belcheri individual

隶属于头索动物亚门的文昌鱼是现存生物中最近似于脊椎动物亚门直接祖先的一个类群,具有重要的进化地位,是研究脊椎动物原始祖先的重要材料和模式动物。随着文昌鱼实验室连续繁殖的成功,全基因组测序成为中国文昌鱼模式化急需完成的工作之一。文章从单条雄性白氏文昌鱼精巢组织中提取高质量的基因组DNA,经EcoRⅠ限制性内切酶和EcoRⅠ甲基化酶酶切,脉冲场电泳选择合适酶切DNA片段,连接线性磷酸化的载体pCC1BAC,转化大肠杆菌EPI300 E.coli,构建了含有44706个克隆的全基因组BAC(Bacterial artificial chromosome)文库,该文库平均插入片段80kb,具有9倍的基因组覆盖率,基本能够满足功能基因等研究需要,为中国文昌鱼全基因组测序打下基础。

As one of the closest living invertebrate relatives of vertebrates, amphioxus （subphylum Cephalochordata） occupies a key position in animal evolution and is becoming the best available proxy and model animal for studying the last common ancestor of all chordates, especially vertebrates. As long-term continuous culturing of amphioxus in laboratory became reliable, for pushing this animal to be a more successful model system, whole-genome sequencing of one or more species derived from this branch will be another urgent issue needed to address. In the present study, we described the construction and characterization of a bacterial artificial chromosome （BAC） library, using a single individual of Chinese amphioxus （Branchiostoma belcheri）. High quality genornic DNA extracted from the spermary was partially digested with EcoR Ⅰ and EcoR Ⅰ methylase. Desirable DNA fragments were isolated by pulsed field gel electrophoresis （PFGE）, ligated to linearized and phosphorylased carrier pCC1BAC, and then transformed to Escherichia coli EPD00. The constructed library consists of 44 706 clones with the average insert fragment size around 80 kb as estimated by PFGE. The representation of the library is about 9 equivalents to the amhioxus genome. These results indicate that the BAC library will be useful for functional genomic studies and facilitate the whole-genome sequencing of Chinese amphioxus