| 尼罗罗非鱼1号染色体分离及文库构建方法研究 |
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| 引用本文： | 杨弘,祝璟琳,邹芝英,李大宇,肖炜,耿青.尼罗罗非鱼1号染色体分离及文库构建方法研究[J].淡水渔业,2018(4):3-8. |
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| 作者姓名： | 杨弘 祝璟琳 邹芝英 李大宇 肖炜 耿青 |
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| 作者单位： | 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 |
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| 基金项目： | 现代农业产业技术体系专项资金资助(CARS-46) |
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| 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。
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| 关 键 词： | 尼罗罗非鱼(Oreochromis niloticus) 1号染色体 显微分离 微克隆 Oreochromis niloticus chromosome 1 microdissection microcloning |
Research on chromosome 1 isolation and genomic library construction of Oreochromis niloticus |
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| YANG Hong,ZHU Jing-ling,ZOU Zhi-ying,LI Da-yu,XIAO Wei,GENG Qing.Research on chromosome 1 isolation and genomic library construction of Oreochromis niloticus[J].Freshwater Fisheries,2018(4):3-8. |
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| Authors: | YANG Hong ZHU Jing-ling ZOU Zhi-ying LI Da-yu XIAO Wei GENG Qing |
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| Abstract: | 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 ampli-fied fragments were detected with developed specific SSR primers.A genome clone of O.niloticus chromosome 1 was con-structed by electrophoresis recycling, transformation and cell cultivation.The constructed micro cloning library of the chro-mosome 1 in O.niloticus contains fragments range from 300 to 600 bp. |
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