尼罗罗非鱼1号染色体分离及文库构建方法研究  

Research on chromosome 1 isolation and genomic library construction of Oreochromis niloticus

作　　者：耿青 肖炜[2] 李大宇[2] 邹芝英[2] 祝璟琳[2] 杨弘[1,2] GENG Qing;XIAO Wei;LI Da-yu;ZOU Zhi-ying;ZHU Jing-ling;YANG Hong(Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214081,Jiaugsu,China;Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization,Ministry of Agriculture;Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,Jiaugsu,Chin)

机构地区：[1]南京农业大学无锡渔业学院,江苏无锡214081 [2]中国水产科学研究院淡水渔业研究中心,农业部淡水渔业和种质资源利用重点实验室,江苏无锡214081

出　　处：《淡水渔业》2018年第4期3-8,共6页Freshwater Fisheries

基　　金：现代农业产业技术体系专项资金资助(CARS-46)

摘　　要：以尼罗罗非鱼(Oreochromis niloticus)为对象,采用体内注射植物血细胞凝集素(PHA)方式收集有丝分裂后期的鱼体头肾细胞,低渗法制片,运用激光显微分离获得尼罗罗非鱼1号染色体,分离后的单染色体通过特异性酶切和LA-PCR体外扩增,得到500~2 000 bp之间的DNA片段,并通过Southern blot和微卫星标记得到验证。扩增片段通过电泳回收、连接转化、感受态细胞培养等步骤构建尼罗罗非鱼1号染色体微克隆文库,克隆片段长度在300~600 bp之间。The head-kidney cells of Oreochromis niloticus were collected by phytohemagglutinin （ PHA） injection in vivo and the metaphase chromosome specimens were prepared by the low osmotic method.The chromosome 1 of O.niloticus was successfully separated by laser microdissection method.Meanwhile,the generated DNA fragments ranging from 500 to 2 000 bp were amplified by specific enzyme cutting and fleetly amplification of linker adaptor mediated PCR （ LA-PCR）.The amplified products of single chromosome were verified by Southern blot and microsatellite markers.SSRs in the amplified fragments were detected with developed specific SSR primers.A genome clone of O.niloticus chromosome 1 was constructed by electrophoresis recycling,transformation and cell cultivation.The constructed micro cloning library of the chromosome 1 in O.niloticus contains fragments range from 300 to 600 bp.

关 键 词：尼罗罗非鱼(Oreochromis niloticus) 1号染色体 显微分离 微克隆 

分 类 号：S917.4[农业科学—水产科学]