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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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 to2 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.
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 to2 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.
引文
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[21]陈学俊,王英,高和琼,等.巴西橡胶树‘热研7-33-97’单染色体显微分离与扩增[J].中国农学通报,2014,30(13):29-33.
[22]Saitoh Y,Ikeda J E.Chromosome microdissection and microcloning[J].Chrom Res,1997,5(2):77-80.
[23]Yan F,Li Z F.New developments in application of laser capture microdissection[J].Chin J Cancer,2004,23(7):860-864.
[24]焦天雷,谢小芳,童治军,等.烟草单染色体的分离与扩增及其SSR的检测[J].福建农林大学学报,2012,41(1):58-62.
[2]Matthew A C,William J.A high quality assembly of the Nile Tilapia(Oreochromis niloticus)genome reveals the structure of two sex determination regions[J].BMC Genom,2017,18:341
[3]Lee B Y,Coutanceau J P,Ozouf C C,et al.Genetic and physical mapping of sex-linked AFLP markers in Nile tilapia(Oreochromis niloticus)[J].Mar Biotechnol,2011,13(3):557-562.
[4]Kocher T D,Lee W J,Sobolewska H,et al.A genetic linkage map of a cichlid fish,the tilapia(Oreochromis niloticus)[J].Genetics,1998,148:1225-1232.
[5]董在杰,缪为民,袁新华,等.尼罗罗非鱼六个性别相关标记的FISH分析[J].中国水产科学,2006,13(4):525-529.
[6]Scalenghe F,Turco E,Edstrom J E,et al.Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes[J].Chromosoma,1981,82(2):205-216.
[7]Liu X,Wang H,Li Y,et al.Preparation of single chromosome for construction of a DNA library using a laser microbeam trap[J].J Biotechnol,2004,109(3):217-261.
[8]Bugno M,Zyga A,Slota E.Microdissected bovine X chromosome segment delineates homologous chromosomal regions in sheep and goat[J].Folia Biol,2008,56(34):149-151.
[9]Hadano S,Watanabe M,Yokoi H,et al.Laser microdissection and single unique primer PCR allow generation of regional chromosome DNA clones from s single human chromosomal[J].Genomics,1991,11(2):364-373.
[10]王兰岚,宋桂英,徐正,等.激光微束切割小冰麦异附加系染色体的研究[J].遗传学报,1997,24(3):238-240.
[11]王槐,宋桂英,王兰岚,等.激光微束切割大麦染色体及染色体片段的分离[J].中国激光,1998,25(7):657-660.
[12]高居荣,彭仁海,韩秀兰,等.激光显微切割技术切割棉花单条染色体的研究[J].实验技术与管理,2009,26(12):32-37.
[13]Yang H Y,Wang J,Kang X Y.Meiotic chromosome preparation techniques of pollen mother cells for laser micro-dissection in Populus spp[J].For Ecosyst,2010,12(2):74-78.
[14]刘志红,俎红丽,赵濛等.山羊单条染色体激光显微切割的研究[J].西北农业学报,2014,23(11):8-11.
[15]Newell P C.Phytohemagglutinin:an initiator of mitosis in cultures of normal human leukocytes[J].Cancer Res,1960,20(7):462-466.
[16]曹丽萍,丁炜东.半微量全血培养法制备罗非鱼染色体标本的条件探索[J].上海水产大学学报,2005,14(4):457-459.
[17]Chen Q,Armstrong K.Characterization of a library from a single microdissected oat(Avena sativa L.)chromosome[J].Genome,1995,38(4):706-714.
[18]朱华平,卢迈新,黄樟翰,等.橙色莫桑比克罗非鱼和荷那龙罗非鱼的染色体核型分析[J].淡水渔业,2009,39(5):18-22.
[19]林义浩.快速获得大量鱼类肾细胞中期分裂相的PHA体内注射法[J].水产学报,1982,6(3):201-208.
[20]耿克祥,闫守庆,李冰,等.Con A与PHA在猪染色体制备中的比较[J].河北北方学院学报,2006,22(6):36-39.
[21]陈学俊,王英,高和琼,等.巴西橡胶树‘热研7-33-97’单染色体显微分离与扩增[J].中国农学通报,2014,30(13):29-33.
[22]Saitoh Y,Ikeda J E.Chromosome microdissection and microcloning[J].Chrom Res,1997,5(2):77-80.
[23]Yan F,Li Z F.New developments in application of laser capture microdissection[J].Chin J Cancer,2004,23(7):860-864.
[24]焦天雷,谢小芳,童治军,等.烟草单染色体的分离与扩增及其SSR的检测[J].福建农林大学学报,2012,41(1):58-62.