尼罗罗非鱼微阵列Fosmid基因组文库的构建及基因筛选  

Construction of a microarrayed Fosmid library and its application in gene isolation in the Nile tilapia( Oreochromis niloticus)

作　　者：李明辉[1] 吴风瑞[1] 熊传奇[1] 曾圣[1] 杨世杰[1] 叶凯[1] 蒋汶洮[1] 孙运侣[1] 黄宝锋[1] 魏莹莹[1] 焦静[1] 顾源[1] 王德寿[1] 

机构地区：[1]西南大学生命科学学院,淡水鱼类资源与生殖发育教育部重点实验室,重庆400715

出　　处：《水产学报》2011年第1期27-34,共8页Journal of Fisheries of China

基　　金：农业部“九四八”项目(2010-Z56);国家“八六三”高科技研究发展计划(2007AA10Z165);国家自然科学基金委员会与香港研究资助局联合科研基金(30831160508);国家自然科学基金(3087195,30770272,31030063);教育部博士点基金项目(20090182110008);重庆市科委攻关项目(CSTC,2008AC1016);重庆市自然科学基金项目(CSTC,2008BB1006)

摘　　要：以pCC2FOS为载体构建尼罗罗非鱼Fosmid基因组文库,共挑选115 200个单菌落,保存在300块384孔样品板中,构建4 800个行池、7 200个列池、300个板池和25个超级池,形成微阵列,并对其进行复制备份。该文库插入片段平均长度约为40 kb,覆盖约罗非鱼基因组的3倍。连续传代实验研究表明,文库具有良好的稳定性。由于构建超级池-板池-行、列池三级池系统形成的微阵列,有助于快速、准确、有效地筛选目的基因,仅需77个PCR反应(超级池25+板池12+行池16+列池24)就能筛选到含有目的基因的一个单克隆,解决了普遍存在的文库筛选难题。通过文库尝试筛选18个与性别分化和生长相关基因,均获得2～5个阳性克隆,说明文库具有较好的实用性。Genomic DNA prepared from the Nile tilapia testis was used for Fosmid library construction.It was randomly sheared to-40 kb fragments.End-repair and recovery of the size-fractionated DNA were manipulated according to the manufacturer’s protocols.Ligation of the end-repaired fragments into the Fosmid vector pCC2FOS（Epicentre,USA） was performed by using T4 DNA ligase for 16 h at 4 ℃.Fosmid clones were packaged using MaxPlaxi Lambda packaging extract provided by the kit.Infected EPI300TM-T1R cells were grown at 37 ℃ in solid medium overnight.Well-separated colonies were picked out and transferred into individual wells of 384-well plates,each well with 300 L culture medium.After overnight incubation at 37 ℃,100 L medium was transferred from each of the 24 wells of every row of the 384-well plates into one well of the 96-well plate to construct row-pools.Totally 16 row-pools were constructed for each 384-well plate.Similarly,100 L medium was transferred from each of the 16 wells of every column of the 384 well plates into one well of the 96-well plate to construct column-pools.Totally 24 column-pools were constructed for each 384-well plate.Plate-pools were constructed by pooling the 16 column-pools and 24 row-pools（200 L from each column-and row-pool） from the same 384-well plate.Totally 4 800 column-pools,7 200 row-pools and 300 plate-pools were constructed and arrayed.The library was further arranged into 25 super-pools,each including a mixture of culture medium from 12 plate-pools（500 L from each plate-pool）.Finally,All 384-well plates and constructed pools were replicated,one copy was kept at-80 ℃ for long-term storage,another copy was kept at-20 ℃ for routine use.The constructed tilapia Fosmid library encompasses 115200 clones.Any gene of interest can be screened only by 3 rounds of minimally 77（25 ＋12 ＋40） colony PCR due to the array of super-,plate-,row-and column-pools.Fosmid stability assays were performed for the constructed library with 9 randomly picked clones by serial cul

关 键 词：尼罗罗非鱼 基因组文库 微阵列 基因筛选 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]