双色原位杂交显示海带配子体5SrDNA与45SrDNA的非连锁关系
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摘要
不等鞭毛类(stramenopiles)许多物种的报道认为,5SrRNA基因与其45SrRNA基因存在连锁关系,但通过设计不同引物进行PCR反应的探索,在海带中没有获得连锁的数据。为了从细胞学角度给予直观的证据,本研究基于海带5SrRNA及45SrRNA基因序列特征分析的基础上,利用双色荧光原位杂交(FISH)的技术和原理,以海带5SrRNA与45SrRNA基因的DNA为模板分别合成带有不同生物素荧光标记的探针,然后杂交到海带配子体的染色体上。FISH图片显示,红色标记的18S-5.8S-25S rDNA探针与绿色标记的5SrDNA探针,在同一幅海带配子体的染色体图片上都只出现了一个杂交信号;从大到小排列的核型显示,45SrDNA及5SrDNA分别定位于海带配子体的第23号和第27号染色体上,直观而清晰地表明5SrDNA并没有与45SrDNA连锁。这是首次尝试性地利用双色荧光原位杂交技术探讨藻类rDNA的染色体定位报道,这个结论有助于将FISH技术应用在海带染色体核型分析等研究。
It had been reported that there was a linkage relationship between 5 S r RNA gene and 45 S r RNA gene in many species of the stramenopiles. However, we did not gain linked data in Laminaria after PCR reaction exploring by designing different primers. To present direct evidence cytologically, this study based on sequence feature analysis of 5 S r RNA and 45 S r RNA gene respectively synthesized fluorescent biotin-labelled probes with the DNA of 5 S r RNA and 45 S r RNA gene in Laminaria as the models to hybridize on the chromosomes of Laminaria gametophytes by dual color fluorescent in situ hybridization(FISH). The FISH result demonstrated that both the labeled probes of 18 S-5.8 S-25 S rDNA(red) and 5 S rDNA(green) presented only one hybridization signal on the same chromosome picture of kelp gametophytes; karyotypes ranked from large to small illustrated that 45 S rDNA and 5 S rDNA were located on Chromosome 23 and Chromosomes 27 of Laminaria gametophytes,respectively, which directly and clearly showed that 5 S rDNA had no genetic linkage relation with 45 S rDNA.This paper tried to use dual-color FISH to investigate the chromosome localization of kelp rDNA at first time. The outcome might contribute to the studies on Laminaria chromosome karyotype analysis via FISH.
It had been reported that there was a linkage relationship between 5 S r RNA gene and 45 S r RNA gene in many species of the stramenopiles. However, we did not gain linked data in Laminaria after PCR reaction exploring by designing different primers. To present direct evidence cytologically, this study based on sequence feature analysis of 5 S r RNA and 45 S r RNA gene respectively synthesized fluorescent biotin-labelled probes with the DNA of 5 S r RNA and 45 S r RNA gene in Laminaria as the models to hybridize on the chromosomes of Laminaria gametophytes by dual color fluorescent in situ hybridization(FISH). The FISH result demonstrated that both the labeled probes of 18 S-5.8 S-25 S rDNA(red) and 5 S rDNA(green) presented only one hybridization signal on the same chromosome picture of kelp gametophytes; karyotypes ranked from large to small illustrated that 45 S rDNA and 5 S rDNA were located on Chromosome 23 and Chromosomes 27 of Laminaria gametophytes,respectively, which directly and clearly showed that 5 S rDNA had no genetic linkage relation with 45 S rDNA.This paper tried to use dual-color FISH to investigate the chromosome localization of kelp rDNA at first time. The outcome might contribute to the studies on Laminaria chromosome karyotype analysis via FISH.
引文
Chen G.F.,Liu Y.,Zhang C.Y.,Chao S.M.,Bao Y.Z.,Guang C.W.,Zhong X.,and Dou D.L.,2013a,Development of r RNA-targeted probes for detection of Prorocentrum micans(Dinophyceae)using whole cell in situ hybridization,Journal of Applied Phycology,25(4):1077-1089
Chen G.F.,Wang Q.F.,Zhang C.Y.,Zhang B.Y.,Wang G.C.,Dou D.L.,Xu Z.,and Yan P.S.,2013b,Development of taxonomic r RNA-targeted probes of two harmful algae:Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization,Acta Oceanologica Sinica.,32(2):66-75
Dai C.H.,an d Fang T.C.,1976,Some preliminary observations on the parthenogenesis in Laminaria japonica Aresch,Yichuan Xuebao(J.Genet.Genomics),3(1):32-38(戴继勋,方宗熙,1976,海带孤雌生殖的初步观察,遗传学报,3(1):32-38)
Dai C.H.,and Fang T.C.,1977,The chromosomes of Laminaria japonica Aresch,Yichuan Xuebao(J.Genet.Genomics),4(4):325-328(戴继勋,方宗熙,1977,海带的染色体,遗传学报,4(4):325-328)
De Jong H.,Fr ansz F.P.,and Zabel P.,1999,High resolution FISH in plants techniques and applications,Trends in Plant Science,4(7):258-263
Dhar M.K.,Kaul S.,Friebe B.,and Gill B.S.,2002,Chromosome identification in Plantago ovata Forsk.through C-banding and FISH,Current Science,83(2):150-152
Figueroa R.I.,Cuadrado A.,Stüken A.,Rodríguez F.,and Fraga S.,2014,Ribosomal DNA organization patterns within the dinoflagellate genus Alexandrium as revealed by FISH:life cycle and evolutionary implications,Protist,165(3):343-363
Frello S.,and Heslop-Ha r rison J.S.,2000,Chromosomal variation in Crocus vernus Hill(Iridaceae)investigated by in situ hybridization of r DNA and a tandemly repeated sequence,Annals of Botany,86(2):317-322
Garcia S.,Garnatje T.,and Kovarík A.,2012,Plant r DNAdatabase:ribosomal DNA loci information goes online,Chromosoma,121(4):389-394
Hu Y.J.,and Zhou Z.G.,2001,Extraction of RAPD-friendly DNA from Laminaria japonica(Phaeophyta)after enzymatic dissociation of the frozen sporophyte tissue,Journal of Applied Phycology,13(5):415-422
Jiang J.,and Gill B.S.,2006,Current status and the future of fluorescence in situ hybridization(FISH)in plant genome research,Genome,49(9):1057-1068
Kawai H.,Muto H.,Fujii T.,and Kato A.,1995,A Linked 5Sr RNA gene in Scytosiphon lomentaria(Scytosiphonales,Phaeophyceae),Journal of Phycology,31(2):306-311
Kawai H.,Nakayama T.,Inouye I.,and Kato A.,1997,Linkage of 5S ribosomal DNA to other r DNAs in the chromophytic algae and related taxa,Journal of Phycology,3(3):505-511
Lavania U.C.,1998,Fluorescence in situ hybridization in genome,chromosome and gene identification in plants,Current Science,74(2):126-133
Lewis R.J.,1996,Chromosomes of the brown algae,Phycologia,35(1):19-40
Li B.J.,2008,Analysis on the genetic variation of mariculture populations and preliminary localization of lucus of gametophyte sex trait in Laminaria,Dissertation for Ph.D.,Ocean University of China,Supervisor:Bao Z.M.,pp.11-15(李秉钧,2008,中国养殖海带不同群体的遗传变异分析及海带配子体性别性状初步定位,博士毕业论文,中国海洋大学,导师:包振民,pp.11-15)
Liu L.,2015,Molecular organization and physical mapping of ribosomal RNA genes in the haploid gametophytes of Saccharina japonica,Thesis for M.S.,Shanghai Ocean University,Supervisor:Zhou Z.G.,pp.21-26(刘丽,2015,海带配子体r RNA基因的序列特征与定位分析,硕士毕业论文,上海海洋大学,导师:周志刚,pp.21-26)
Liu Y.,Bi Y.H.,and Zhou Z.G.,2012,Karyological observation on Saccharina japonica chromosomes stained with DAPI,Shuichan Xuebao(J.Fish.Chin.),36(1):50-54(刘宇,毕燕会,周志刚,2012,海带染色体的DAPI染色及核型初步分析,水产学报,36(1):50-54)
Liu Y.,Bi Y.H.,Gu J.G.,Li L.H.,and Zhou Z.G.,2012b,Localization of a female-specific marker on the chromosomes of the brown seaweed Saccharina japonica using fluorescence in situ hybridization,PLo S One,7(11):e48784
Majka J.,Majk a M.,Kwiatek M.,and Wisniewska H.,2017,Similarities and differences in the nuclear genome organization within Pooideae species revealed by comparative genomic in situ hybridization(GISH),J.Appl.Genet.,58(2):151-161
Montijn M.B.,Houtsmuller A.B.,ten Hoopen R.,Oud J.L.,and Nanninga N.,1999,The 5S r RNA gene clusters have a defined orientation toward the nucleolus in Petunina hybrida and Crepis capillaries,Chromosome Research.,7(5):387-399
Pedersen C.,and Linde-Laursen I.,1994,Chromosomal locations of four minor r DNA loci and a marker microsatellite sequence in barley,Chromosome Research.,2(1):65-71
Scaldaferro M.A.,da Cruz M.V.,Cecchini N.M.,and Mos-cone E.A.,2016,FISH and Ag Nor mapping of the 45S and 5Sr RNA genes in wild and cultivated species of Capsicum(Solananceae),Genome,59(2):95-113
Schwarzacher T.,and Heslop-Harrison J.S.,eds.,2000,Practical in situ Hybridization,Oxford,Bios Scientific Publishers,New York,US,pp.203
Thomas H.M.,Harper J.A.,and Morgan W.G.,2001,Gross chromosome rearrangements are occurring in an accession of the grass Lolium rigidum,Chromosome Research,9(7):585-590
Wasko A.P.,and Galetti P.M.J.,2000,Mapping 18S ribosomal genes in fish of the genus Brycon(Characidae)by fluorescence in situ hybridization(FISH),Genetics and Molecular Biology,23(1):135-138
Yabu H.,1973,Alternation of chromosomes in the life history of Laminaria japonica Aresch,Bulletin of the Faculty of Fisheries Hokkaido University,23(4):171-176
Yabu H.,and Yasui H.,1991,Chromosome number in four species of Laminaria(Phaeophyta),Japanese Journal of Phycology,39(2):185-187
Ye N.H.,Zhang X.W.,Miao M.,Fan X.,Zheng Y.,Xu D.,Wang J.F.,Zhou L.,Wang D.S.,Gao Y.,Wang Y.T.,Shi W.Y.,Ji P.F.,Li D.M.,Guan Z.,Shao C.W.,Zhuang Z.M.,Gao Z.Q.,Qi J.,and Zhao F.Q.,2015,Saccharina genomes provide novel insight into kelp biology,Nature Communications,6:6986
Yotsukura N.,Kawai T.,Motomura T.,and Ichimura T.,2002,Tandem 5S ribosomal RNA genes and the spacer region sequences of three Japanese Laminaria species,Journal of Applied Phycology,14(4):233-239
Zhao L.J.,and Li J.J.,2004,Advances in chromosome C-banding and in situ hybridization of plants,Harbing Shifan Daxue Ziran Kexue Xuebao(Natural Sciences Journal of Harbin Normal University),20(5):86-88(赵丽娟,李集临,2004,植物染色体C分带和原杂交的研究应用,哈尔滨师范大学自然科学学报,20(5):86-88)
Zhou L.R.,Dai J.X.,and Shen S.D.,2004,An improved chromosome preparation from male gametophyte of Laminaria japonica(Heterokontophyta),Hydrobiologia,512(1-3):141-144
Chen G.F.,Wang Q.F.,Zhang C.Y.,Zhang B.Y.,Wang G.C.,Dou D.L.,Xu Z.,and Yan P.S.,2013b,Development of taxonomic r RNA-targeted probes of two harmful algae:Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization,Acta Oceanologica Sinica.,32(2):66-75
Dai C.H.,an d Fang T.C.,1976,Some preliminary observations on the parthenogenesis in Laminaria japonica Aresch,Yichuan Xuebao(J.Genet.Genomics),3(1):32-38(戴继勋,方宗熙,1976,海带孤雌生殖的初步观察,遗传学报,3(1):32-38)
Dai C.H.,and Fang T.C.,1977,The chromosomes of Laminaria japonica Aresch,Yichuan Xuebao(J.Genet.Genomics),4(4):325-328(戴继勋,方宗熙,1977,海带的染色体,遗传学报,4(4):325-328)
De Jong H.,Fr ansz F.P.,and Zabel P.,1999,High resolution FISH in plants techniques and applications,Trends in Plant Science,4(7):258-263
Dhar M.K.,Kaul S.,Friebe B.,and Gill B.S.,2002,Chromosome identification in Plantago ovata Forsk.through C-banding and FISH,Current Science,83(2):150-152
Figueroa R.I.,Cuadrado A.,Stüken A.,Rodríguez F.,and Fraga S.,2014,Ribosomal DNA organization patterns within the dinoflagellate genus Alexandrium as revealed by FISH:life cycle and evolutionary implications,Protist,165(3):343-363
Frello S.,and Heslop-Ha r rison J.S.,2000,Chromosomal variation in Crocus vernus Hill(Iridaceae)investigated by in situ hybridization of r DNA and a tandemly repeated sequence,Annals of Botany,86(2):317-322
Garcia S.,Garnatje T.,and Kovarík A.,2012,Plant r DNAdatabase:ribosomal DNA loci information goes online,Chromosoma,121(4):389-394
Hu Y.J.,and Zhou Z.G.,2001,Extraction of RAPD-friendly DNA from Laminaria japonica(Phaeophyta)after enzymatic dissociation of the frozen sporophyte tissue,Journal of Applied Phycology,13(5):415-422
Jiang J.,and Gill B.S.,2006,Current status and the future of fluorescence in situ hybridization(FISH)in plant genome research,Genome,49(9):1057-1068
Kawai H.,Muto H.,Fujii T.,and Kato A.,1995,A Linked 5Sr RNA gene in Scytosiphon lomentaria(Scytosiphonales,Phaeophyceae),Journal of Phycology,31(2):306-311
Kawai H.,Nakayama T.,Inouye I.,and Kato A.,1997,Linkage of 5S ribosomal DNA to other r DNAs in the chromophytic algae and related taxa,Journal of Phycology,3(3):505-511
Lavania U.C.,1998,Fluorescence in situ hybridization in genome,chromosome and gene identification in plants,Current Science,74(2):126-133
Lewis R.J.,1996,Chromosomes of the brown algae,Phycologia,35(1):19-40
Li B.J.,2008,Analysis on the genetic variation of mariculture populations and preliminary localization of lucus of gametophyte sex trait in Laminaria,Dissertation for Ph.D.,Ocean University of China,Supervisor:Bao Z.M.,pp.11-15(李秉钧,2008,中国养殖海带不同群体的遗传变异分析及海带配子体性别性状初步定位,博士毕业论文,中国海洋大学,导师:包振民,pp.11-15)
Liu L.,2015,Molecular organization and physical mapping of ribosomal RNA genes in the haploid gametophytes of Saccharina japonica,Thesis for M.S.,Shanghai Ocean University,Supervisor:Zhou Z.G.,pp.21-26(刘丽,2015,海带配子体r RNA基因的序列特征与定位分析,硕士毕业论文,上海海洋大学,导师:周志刚,pp.21-26)
Liu Y.,Bi Y.H.,and Zhou Z.G.,2012,Karyological observation on Saccharina japonica chromosomes stained with DAPI,Shuichan Xuebao(J.Fish.Chin.),36(1):50-54(刘宇,毕燕会,周志刚,2012,海带染色体的DAPI染色及核型初步分析,水产学报,36(1):50-54)
Liu Y.,Bi Y.H.,Gu J.G.,Li L.H.,and Zhou Z.G.,2012b,Localization of a female-specific marker on the chromosomes of the brown seaweed Saccharina japonica using fluorescence in situ hybridization,PLo S One,7(11):e48784
Majka J.,Majk a M.,Kwiatek M.,and Wisniewska H.,2017,Similarities and differences in the nuclear genome organization within Pooideae species revealed by comparative genomic in situ hybridization(GISH),J.Appl.Genet.,58(2):151-161
Montijn M.B.,Houtsmuller A.B.,ten Hoopen R.,Oud J.L.,and Nanninga N.,1999,The 5S r RNA gene clusters have a defined orientation toward the nucleolus in Petunina hybrida and Crepis capillaries,Chromosome Research.,7(5):387-399
Pedersen C.,and Linde-Laursen I.,1994,Chromosomal locations of four minor r DNA loci and a marker microsatellite sequence in barley,Chromosome Research.,2(1):65-71
Scaldaferro M.A.,da Cruz M.V.,Cecchini N.M.,and Mos-cone E.A.,2016,FISH and Ag Nor mapping of the 45S and 5Sr RNA genes in wild and cultivated species of Capsicum(Solananceae),Genome,59(2):95-113
Schwarzacher T.,and Heslop-Harrison J.S.,eds.,2000,Practical in situ Hybridization,Oxford,Bios Scientific Publishers,New York,US,pp.203
Thomas H.M.,Harper J.A.,and Morgan W.G.,2001,Gross chromosome rearrangements are occurring in an accession of the grass Lolium rigidum,Chromosome Research,9(7):585-590
Wasko A.P.,and Galetti P.M.J.,2000,Mapping 18S ribosomal genes in fish of the genus Brycon(Characidae)by fluorescence in situ hybridization(FISH),Genetics and Molecular Biology,23(1):135-138
Yabu H.,1973,Alternation of chromosomes in the life history of Laminaria japonica Aresch,Bulletin of the Faculty of Fisheries Hokkaido University,23(4):171-176
Yabu H.,and Yasui H.,1991,Chromosome number in four species of Laminaria(Phaeophyta),Japanese Journal of Phycology,39(2):185-187
Ye N.H.,Zhang X.W.,Miao M.,Fan X.,Zheng Y.,Xu D.,Wang J.F.,Zhou L.,Wang D.S.,Gao Y.,Wang Y.T.,Shi W.Y.,Ji P.F.,Li D.M.,Guan Z.,Shao C.W.,Zhuang Z.M.,Gao Z.Q.,Qi J.,and Zhao F.Q.,2015,Saccharina genomes provide novel insight into kelp biology,Nature Communications,6:6986
Yotsukura N.,Kawai T.,Motomura T.,and Ichimura T.,2002,Tandem 5S ribosomal RNA genes and the spacer region sequences of three Japanese Laminaria species,Journal of Applied Phycology,14(4):233-239
Zhao L.J.,and Li J.J.,2004,Advances in chromosome C-banding and in situ hybridization of plants,Harbing Shifan Daxue Ziran Kexue Xuebao(Natural Sciences Journal of Harbin Normal University),20(5):86-88(赵丽娟,李集临,2004,植物染色体C分带和原杂交的研究应用,哈尔滨师范大学自然科学学报,20(5):86-88)
Zhou L.R.,Dai J.X.,and Shen S.D.,2004,An improved chromosome preparation from male gametophyte of Laminaria japonica(Heterokontophyta),Hydrobiologia,512(1-3):141-144