用于目标序列染色体定位的镜鲤BAC-FISH体系构建
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摘要
为了构建用于镜鲤(Cyprinus carpio var.specularis)特定基因组序列染色体定位的实验体系,在细菌人工染色体(Bacterial Artificial Chromosome,BAC)文库筛选池中对已知短序列基因组片段进行PCR扩增,筛选出包含目标序列的BAC克隆,提取BAC质粒进行缺刻平移标记制备探针,开展荧光原位杂交(Fluorescencein situ hybridization,FISH)实验。通过对染色体片前处理、BAC质粒探针制备、C0t-1 DNA封闭基因组重复序列、预杂交、荧光染料选择、信号放大等一系列实验条件和方法的探索优化,成功实现了目标序列在镜鲤有丝分裂中期染色体上的定位。定位对象既包括在染色体上有单一位点的序列,如斑马鱼微卫星标记Z6884和Z4268,也包括在染色体上有多个位点的重复序列,如黄河鲤性别相关标记CCmf1。来自斑马鱼同一条染色体上的两个微卫星标记被分别定位于镜鲤不同染色体上,为鲤鱼染色体数目加倍的进化假设提供了一项直接实验证据,同时将现有遗传连锁图谱与染色体对应起来,可作为染色体识别和细胞遗传学图谱构建的依据。黄河鲤性别相关重复序列被定位于不少于四条染色体上,为性别决定相关基因的筛查提供了研究线索。这一BAC-FISH实验体系将成为鲤细胞遗传学图谱构建、基因组进化和比较基因组学研究中的重要研究工具。
An experimental system has been constructed for localizing specific genomic sequences on mirror carp(Cyprinus carpio var.specularis) chromosomes.In order to obtain individual BAC clones containing the target sequences,short genomic sequences were amplified by PCR in bacterial artificial chromosome(BAC) screening pools.BAC plasmid DNA was isolated and labeled by nick translation to prepare fluorescence in situ hybridization(FISH) probes.Via optimizing a series of protocols for chromosome slides pre-treatment,BAC plasmid probe preparation,repetitive sequences blocking by C0t-1 DNA,pre-hybridization,candidate fluorescence dyes evaluation,signal amplification and so on,the BAC-FISH system has been successfully established for mirror carp metaphase chromosomes,which is able to localize both single-copy markers and repetitive sequences.As supporting evidence to the genome duplication hypothesis of carp evolution,two microsatellite markers from zebrafish chromosome 17,namely Z6884 and Z4268,have been localized on two separate mirror carp chromosomes,with one marker on each chromosome.A male-specific marker CCmf1 from Yellow River carp has been localized on at least four chromosomes of mirror carp,suggesting that it might be a repetitive sequence useful for sex determining gene searching.The established mirror carp BAC-FISH experimental system would serve as a powerful tool for carp cytogenetic mapping,genome evolution and comparative genomics research.
An experimental system has been constructed for localizing specific genomic sequences on mirror carp(Cyprinus carpio var.specularis) chromosomes.In order to obtain individual BAC clones containing the target sequences,short genomic sequences were amplified by PCR in bacterial artificial chromosome(BAC) screening pools.BAC plasmid DNA was isolated and labeled by nick translation to prepare fluorescence in situ hybridization(FISH) probes.Via optimizing a series of protocols for chromosome slides pre-treatment,BAC plasmid probe preparation,repetitive sequences blocking by C0t-1 DNA,pre-hybridization,candidate fluorescence dyes evaluation,signal amplification and so on,the BAC-FISH system has been successfully established for mirror carp metaphase chromosomes,which is able to localize both single-copy markers and repetitive sequences.As supporting evidence to the genome duplication hypothesis of carp evolution,two microsatellite markers from zebrafish chromosome 17,namely Z6884 and Z4268,have been localized on two separate mirror carp chromosomes,with one marker on each chromosome.A male-specific marker CCmf1 from Yellow River carp has been localized on at least four chromosomes of mirror carp,suggesting that it might be a repetitive sequence useful for sex determining gene searching.The established mirror carp BAC-FISH experimental system would serve as a powerful tool for carp cytogenetic mapping,genome evolution and comparative genomics research.
引文
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[24]Huan P,Zhang X,Li F,et al.Chromosomal localization of5S rDNA in Chinese shrimp(Fenneropenaeus chinensis):achromosome-specific marker for chromosome identification[J].Chinese Journal of Oceanology and Limnology,2010,28(2):233—238
[25]Yu X J,Zhou T,Li Y C,et al.Chromosomes of ChineseFresh-water Fishes[M].Beijing:Science Press.1989,13[余先觉,周暾,李渝成,等.中国淡水鱼类染色体.北京:科学出版社.1989,13]
[26]David L,Shula B,Marcus W F,et al.Recent duplication ofthe common carp(Cyprinus carpio L.)Genome as revealedby analyses of microsatellite loci[J].Molecular BiologyEvolution,2003,20(9):1425—1434
[27]Sun X,Liang L.A genetic linkage map of common carp(Cyprinus carpio L.)and mapping of a locus associated withcold tolerance[J].Aquaculture,2004,238(1—4):165—172
[28]Chen J,Wang Y,Yue Y,et al.A novel male-specific DNAsequence in the common carp,Cyprinus carpio[J].Molecu-lar and Cellular Probes,2009,23(5):235—239
[29]Charlesworth D,Charlesworth B,Marais G.Steps in theevolution of heteromorphic sex chromosomes[J].Heredity,2005,95(2):118—128
[2]Koo D H,Jo S H,Bang J W,et al.Integration of cytogeneticand genetic linkage maps unveils the physical architecture oftomato chromosome 2[J].Genetics,2008,179(3):1211—1220
[3]Luo M C,Ma Y,You F M,et al.Feasibility of physical mapconstruction from fingerprinted bacterial artificial chromo-some libraries of polyploid plant species[J].BMC Genomics,2010,11:122
[4]Schwarzacher T,Leitch A R,Bennett M D,et al.In situ lo-calization of parental genomes in a wide hybrid[J].Annalsof Botany,1989,64(3):315—324
[5]Leitch A R,Mosg ller W,Schwarzacher T,et al.Genomic insitu hybridization to sectioned nuclei shows chromosomedomains in grass hybrids[J].Journal of Cell Science,1990,95(3):335—341
[6]Han Y H,Zhang Z H,Liu J H,et al.Distribution of the tan-dem repeat sequences and karyotyping in cucumber(Cucu-mis sativus L.)by fluorescence in situ hybridization[J].Cy-togenetic and Genome Research,2008,122(1):80—88
[7]Giannuzzi G,D'Addabbo P,Gasparro M,et al.Analysis ofhigh-identity segmental duplications in the grapevine ge-nome[J].BMC Genomics,2011,12:436
[8]Salvo-Garrido H,S Travella,L J Bilham,et al.The distribu-tion of transgene insertion sites in barley determined byphysical and genetic mapping[J].Genetics,2004,167(3):1371—1379
[9]Phillips R B,Amores A,Morasch M R,et al.Assignment ofzebrafish genetic linkage groups to chromosomes[J].Cyto-genetic and Genome Research,2006,114(2):155—162
[10]Freeman J L,Adeniyi A,Banerjee R,et al.Definition of thezebrafish genome using flow cytometry and cytogeneticmapping[J].BMC Genomics,2007,8:195
[11]Alcivar-Warren A,Meehan-Meola D,Wang Y,et al.Isolationand mapping of telomeric pentanucleotide(TAACC)n re-peats of the Pacific Whiteleg Shrimp,Penaeus vannamei,using fluorescence in situ hybridization[J].Marine Biotech-nology,2006,8(5):467—480
[12]Singh M,Kumar R,Nagpure N S,et al.Chromosomal local-ization of 18S and 5S rDNA using FISH in the genus Tor(Pisces,Cyprinidae)[J].Genetica,2009,137(3):245—252
[13]Huan P,Zhang X,Li F,et al.Chromosomal localization andmolecular marker development of the lipopolysaccharide andbeta-1,3-glucan binding protein gene in the Zhikong scallopChlamys farreri(Jones et Preston)(Pectinoida,Pectinidae)[J].Genetics and Molecular Biology,2010,33(1):36—43
[14]LüZ M,Yang A G,Wang Q Y,et al.Preliminary study onfish identification of chromosomes in hybrids from crossbetween Patinopecten yessoensis and Chlamys farreri[J].Acta Hydrobiologica Sinica,2010,34(4):709—715[吕振明,杨爱国,王清印,等.FISH技术在栉孔扇贝和虾夷扇贝杂交子代染色体识别中的运用初探.水生生物学报,2010,34(4):709—715
[15]Shizuya H,Birren B,Kim U J,et al.Cloning and stablemaintenance of 300-kilobase-pair fragments of human DNAin Escherichia coli using an F-factor-based vector[J].Pro-ceedings of the National Academy of Sciences of the UnitedStates of America,1992,89(18):8794—8797
[16]Zhang X J,Huan P.Fluorescence in situ hybridization map-ping[A].In:Sun X W,Xu P(Eds.),Technologies and Re-search Progresses in Aquatic Genomics[C].Beijing:OceanPress.2011,62—86[张晓军,郇聘.荧光原位杂交作图.见:孙效文,徐鹏.水产基因组技术与研究进展.北京:海洋出版社.2011,62—86
[17]Huan P.Application of Fluorescence in situ hybridization(FISH)in Chinese shrimp,Fenneropenaeus chinensis andZhikong scallop,Chlamys farreri:Gene mapping and mapintegration[D].Thesis for PhD of Science.Institute ofOceanology,Chinese Academy of Sciences,Qingdao.2009[郇聘.利用荧光原位杂交技术(FISH)对中国明对虾和栉孔扇贝若干重要基因定位的研究.博士学位论文,中国科学院海洋研究所,青岛.2009]
[18]Li Y,Xu P,Zhao Z,et al.Construction and characterizationof the bac library for common carp Cyprinus carpio L.andestablishment of microsynteny with zebrafish Danio rerio[J].Marine Biotechnology,2011,13(4):706—712
[19]Spector D L,Goldman R D,Leinwand L A.A LaboratoryManual[M].New York:Cold Spring Harbor LaboratoryPress.1998,111.26—111.36
[20]Zwick M S,Hanson R E,Islam-Faridi M N,et al.A rapidprocedure for the isolation of C0t-1 DNA from plants[J].Genome,1997,40(1):138—142
[21]Shoguchi E,Kawashima T,Satou Y,et al.Chromosomalmapping of 170 BAC clones in the ascidian Ciona intesti-nalis[J].Genome Research,2006,16(2):297—303
[22]Wang K,Zhang Y J,Guan B,et al.Fluorescence in situ hy-bridization of bacterial artificial chromosome in cotton[J].Progress in Biochemistry and Biophysics,2007,34(11):1216—1222[王凯,张燕洁,关兵,等.棉花细菌人工染色体的荧光原位杂交(BAC-FISH)技术.生物化学与生物物理进展,2007,34(11):1216—1222]
[23]Huang X T.Cytogenetic characterization of scallop chromo-somes[D].Thesis for PhD of Science.Ocean University ofChina,Qingdao.2009[黄晓婷.扇贝染色体的细胞遗传学研究.博士学位论文,中国海洋大学,青岛.2009]
[24]Huan P,Zhang X,Li F,et al.Chromosomal localization of5S rDNA in Chinese shrimp(Fenneropenaeus chinensis):achromosome-specific marker for chromosome identification[J].Chinese Journal of Oceanology and Limnology,2010,28(2):233—238
[25]Yu X J,Zhou T,Li Y C,et al.Chromosomes of ChineseFresh-water Fishes[M].Beijing:Science Press.1989,13[余先觉,周暾,李渝成,等.中国淡水鱼类染色体.北京:科学出版社.1989,13]
[26]David L,Shula B,Marcus W F,et al.Recent duplication ofthe common carp(Cyprinus carpio L.)Genome as revealedby analyses of microsatellite loci[J].Molecular BiologyEvolution,2003,20(9):1425—1434
[27]Sun X,Liang L.A genetic linkage map of common carp(Cyprinus carpio L.)and mapping of a locus associated withcold tolerance[J].Aquaculture,2004,238(1—4):165—172
[28]Chen J,Wang Y,Yue Y,et al.A novel male-specific DNAsequence in the common carp,Cyprinus carpio[J].Molecu-lar and Cellular Probes,2009,23(5):235—239
[29]Charlesworth D,Charlesworth B,Marais G.Steps in theevolution of heteromorphic sex chromosomes[J].Heredity,2005,95(2):118—128