橘色双冠丽鱼黑素皮质素受体1基因(MC1R)整胚原位杂交
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摘要
为探究黑素皮质素受体1基因(MC1R)在橘色双冠丽鱼胚胎发育和体色形成过程中的表达定位及功能,本研究首先制备了MC1R基因的RNA正反义探针,T7方向转录的正义RNA探针质量浓度为447.529 ng/μL,SP6方向转录的反义RNA探针质量浓度为342.698 ng/μL。经10～20倍稀释后的探针用于原位杂交,MC1R基因探针表达定位显示,随橘色双冠丽鱼胚胎的发育杂交信号总体呈逐渐减弱趋势,原肠期和视泡期其卵黄和胚体侧卧部分有杂交信号分布;出膜期其脊柱、卵黄囊及其内容物和色素细胞内出现杂交信号;出膜期第1天,卵黄表面有信号分布。总体来看,杂交信号在脊索、卵黄囊、卵黄囊内容物质及色素细胞有特异表达,而以正义探针作为阴性对照组在5个胚胎阶段均无任何信号,杂交信号显现的MC1R表达定位说明其在橘色双冠丽鱼色素细胞的分化、迁移中起到重要调控作用,也与神经、营养、免疫功能相关,初步建立了鱼类体色相关基因功能和定位研究的整胚原位杂交方法。
In order to explore the melanocortin receptor 1 gene(MC1R) in Amphilophus citrinellus embryo development and the formation of the body color expression location and function of the test preparation of MC1R gene RNA is antisense probes, T7 has sense direction of transcription of RNA probe concentration is 447.529 ng/μL,SP6 direction of transcription antisense RNA probe concentration is 342.698 ng/μL. After 10 to 20 times dilution probe was used for in situ hybridization, the MC1R gene probe expression positioning shows that A. citrinellus embryo developmental hybridization signal was weakening trend in general, the intestinal phase and bubble phase,yolk and lateral embryonic body parts have a hybridization signal distribution; in the film stage, hybridization signals appeared in the spine, yolk sac and yolk sac contents and pigment cells. There is a signal distribution on the yolk surface. In general, hybridization signals are specifically expressed in chordate, yolk sac and yolk sac contents and pigment cells, and the sense probe as a negative control group in five embryonic stages without any signal,sage MC1R expression positioning shows that the signal is revealed in A. citrinellus pigment cells that play an important regulating role in differentiation, migration, and are also associated with nerve, nutrition, immune function, preliminary establishment of fish body color related gene function and orientation of the entire embryo in situ hybridization method.
In order to explore the melanocortin receptor 1 gene(MC1R) in Amphilophus citrinellus embryo development and the formation of the body color expression location and function of the test preparation of MC1R gene RNA is antisense probes, T7 has sense direction of transcription of RNA probe concentration is 447.529 ng/μL,SP6 direction of transcription antisense RNA probe concentration is 342.698 ng/μL. After 10 to 20 times dilution probe was used for in situ hybridization, the MC1R gene probe expression positioning shows that A. citrinellus embryo developmental hybridization signal was weakening trend in general, the intestinal phase and bubble phase,yolk and lateral embryonic body parts have a hybridization signal distribution; in the film stage, hybridization signals appeared in the spine, yolk sac and yolk sac contents and pigment cells. There is a signal distribution on the yolk surface. In general, hybridization signals are specifically expressed in chordate, yolk sac and yolk sac contents and pigment cells, and the sense probe as a negative control group in five embryonic stages without any signal,sage MC1R expression positioning shows that the signal is revealed in A. citrinellus pigment cells that play an important regulating role in differentiation, migration, and are also associated with nerve, nutrition, immune function, preliminary establishment of fish body color related gene function and orientation of the entire embryo in situ hybridization method.
引文
[1]Gall J G,Pardue M L.Formation and detection of RNA-DNA hybrid molecules in cytological preparations[J].Proceedings of the National Academy of Sciences of the United States of America,1969,69(2):378-383.
[2]Westerfield M.The zebrafish book:a guide for the laboratory use of zebrafish(Danio rerio)[M].5th ed.Eugene:University of Oregon Press,2007.
[3]杨冰洁.几种紫薇属植物染色体制片技术优化及原位杂交体系的建立[D].北京:北京林业大学,2013.Yang B J.Optimization of chromosome sectioning and establishment of FISH and GISH of Lagerstroemia L.[D].Beijing:Beijing Forestry University,2013(in Chinese).
[4]鲁翠云,张明昭,李超,等.荧光原位杂交技术在鱼类基因定位研究中的应用[J].水产学杂志,2013,26(4):51-57.Lu C Y,Zhang M Z,Li C,et al.Application of fluorescence in situ hybridization(FISH)techniques in gene location in fish[J].Chinese Journal of Fisheries,2013,26(4):51-57(in Chinese).
[5]Cao J M,Li S Q,Zhang H W,et al.High mobility group B proteins regulate mesoderm formation and dorsoventral patterning during zebrafish and Xenopus early development[J].Mechanisms of Development,2012,129(9-12):263-274.
[6]Lauter G,S?ll I,Hauptmann G.Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain[J].Neural Development,2011,6:10.
[7]陈琼霞,镇鸿燕,李艳,等.基于石蜡包埋组织荧光原位杂交的技术问题[J].中国医药指南,2013,11(13):382.Chen Q X,Zhen H Y,Li Y,et al.Based on fluorescence in situ hybridization of paraffin-embedded tissue[J].Guide of China Medicine,2013,11(13):382(in Chinese).
[8]靳雁斌,丁学锋,司慧远,等.小鼠Cpne5基因mRNA水平的表达分布及其在胚胎的表达定位[J].标记免疫分析与临床,2013,20(2):93-96.Jin Y B,Ding X F,Si H Y,et al.Expression of mouse Cpne5 gene mRNA in various tissues and its location in embryo[J].Labeled Immunoassays and Clinical Medicine,2013,20(2):93-96(in Chinese).
[9]蒋丽,王阳阳,程安达,等.鲤鱼(Cyprinus carpio)外异蛋白A受体Edar基因的克隆及表达定位[J].生物技术通报,2013(12):99-107.Jiang L,Wang Y Y,Cheng A D,et al.Preliminary study of Edar gene cloning and localization of expression in Cyprinus carpio[J].Biotechnology Bulletin,2013(12):99-107(in Chinese).
[10]叶寒青,陈松林,刘洋,等.表达绿色荧光蛋白基因的花鲈胚胎干细胞株的建立及其体外分化[J].高技术通讯,2006,16(1):61-66.Ye H Q,Chen S L,Liu Y,et al.Establishment and differentiation in vitro of sea perch embryonic stem cell line expressing GFP gene[J].Chinese High Technology Letters,2006,16(1):61-66(in Chinese).
[11]曹建萌,卢迈新,叶星,等.尼罗罗非鱼整胚原位杂交技术的建立和初步应用[J].水产学报,2014,38(11):1847-1854.Cao J M,Lu M X,Ye X,et al.A novel protocol of whole mount in situ hybridization(WISH)and its primary application in Nile tilapia[J].Journal of Fisheries of China,2014,38(11):1847-1854(in Chinese).
[12]杨永升,李宁,邓学梅,等.黑素皮质素受体1:哺乳动物黑色素形成中的关键基因[J].遗传,2004,26(4):544-550.Yang Y S,Li N,Deng X H,et al.MC1R:the key gene in mammalian melanin synthesis[J].Hereditas(Beijing),2004,26(4):544-550(in Chinese).
[13]FitzGerald L M,Fryer J L,Dwyer T,et al.Effect of MELANOTAN?,[Nle4,D-Phe7]-α-MSH,on melanin synthesis in humans with MC1R variant alleles[J].Peptides,2006,27(2):388-394.
[14]Adalsteinsson S,Hersteinsson P,Gunnarsson E.Fox colors in relation to colors in mice and sheep[J].Journal of Heredity,1987,78(4):235-237.
[15]孙静.红褐色突变貉毛色基因MC1R的序列分析及其表达水平的研究[D].青岛:青岛农业大学,2012.Sun J.Sequence analysis and expression level study on MC1R gene in reddish-brown raccoon dog[D].Qingdao:Qingdao Agricultural University,2012(in Chinese).
[16]任玉红,杨刚,范瑞文,等.黑素皮质素受体1(MC1R)在不同毛色羊驼皮肤组织中的表达与定位研究[J].畜牧兽医学报,2012,43(7):1049-1055.Ren Y H,Yang G,Fan R W,et al.Expression and immunolocalization of melanocortin receptor 1(MC1R)in alpaca skin with different coat color[J].Acta Veterinaria et Zootechnica Sinica,2012,43(7):1049-1055(in Chinese).
[17]胡建尊,李康乐,项松平,等.瓯江彩鲤体色调控相关因子MC1R的克隆与表达分析[J].上海海洋大学学报,2013,22(4):518-523.Hu J Z,Li K L,Xiang S P,et al.Cloning and expression analysis of color-related regulation factor MC1R in Oujiang color common carp[J].Journal of Shanghai Ocean University,2013,22(4):518-523(in Chinese).
[18]蒋燕玲.橘色双冠丽鱼体色发育变化及体色相关基因TYR的克隆与表达研究[D].上海:上海海洋大学,2016.Jiang Y L.Body color variation and cloning,expression analysis of TYR gene in Amphilophus citrinellus[D].Shanghai:Shanghai Ocean University,2016(in Chinese).
[19]孙桂金,潘杰,刘可春,等.苯硫脲对斑马鱼黑色素生成及早期发育的影响[J].水产科学,2011,30(7):387-390.Sun G J,Pan J,Liu K C,et al.Effects of N-phenylthiourea(PTU)on melanogenesis and early development in zebrafish(Danio rerio)[J].Fisheries Science,2011,30(7):387-390(in Chinese).
[20]徐永江,朱学武,柳学周,等.半滑舌鳎(Cynoglossus semilaevis)黑色素富集激素基因的克隆和表达[J].渔业科学进展,2017,38(1):81-90.Xu Y J,Zhu X B,Liu X Z,et al.Cloning and expression of melanin-concentrating hormone in half-smooth tongue sole(Cynoglossus semilaevis)[J].Progress in Fishery Sciences,2017,38(1):81-90(in Chinese).
[21]王东,方军,厉有名,等.链霉蛋白酶提高胃镜检查图像可见度的前瞻性、多中心、双盲、随机对照临床研究[J].中华消化内镜杂志,2013,30(11):604-607.Wang D,Fang J,Li Y M,et al.Effectiveness of premedication with pronase for visualization of the mucosa during endoscopy:a double-blinded,multicenter,prospective,randomized study[J].Chinese Journal of Digestive Endoscopy,2013,30(11):604-607(in Chinese).
[22]纪莉莎,管泱,姜昕.链霉蛋白酶在胃镜检查中的临床应用[J].中国继续医学教育,2015,7(23):129-130.Ji L S,Guan Y,Jiang X.Application of pronase in gastroscopy examination[J].China Continuing Medical Education,2015,7(23):129-130(in Chinese).
[23]樊竑冶,彭忠禄,谢华平,等.斑马鱼整体原位杂交的技术改良[J].激光生物学报,2010,19(1):115-119.Fan H Y,Peng Z L,Xie H P,et al.Improvement of whole-mount in situ hybridization to zebrafish embryos[J].Acta Laser Biology Sinica,2010,19(1):115-119(in Chinese).
[24]张春霞,刘峰.斑马鱼高分辨率整胚原位杂交实验方法与流程[J].遗传,2013,35(4):522-528.Zhang C X,Liu F.A brief protocol for high-resolution whole mount in situ hybridization in zebrafish[J].Hereditas(Beijing),2013,35(4):522-528(in Chinese).
[25]韦敏侠,宋红梅,祁宝伦,等.橘色双冠丽鱼胚后色素细胞发育与体色变化[J].上海海洋大学学报,2015,24(1):28-35.Wei M X,Song H M,Qi B L,et al.Pigment cells development and body color variation of postembryonic development in Amphilophus citrinellus(Günther1864)[J].Journal of Shanghai Ocean University,2015,24(1):28-35(in Chinese).
[26]程炜轩,许国焕,熊达,等.黄颡鱼黑色素细胞原代培养及迁移相关基因克隆分析[J].生态毒理学报,2014,9(6):1035-1040.Cheng W X,Xu G H,Xiong D,et al.Primary culturing of melanoma cells and cloning analysis of migrationrelated gene in Pelteobagrus fulvidraco[J].Asian Journal of Ecotoxicology,2014,9(6):1035-1040(in Chinese).
[2]Westerfield M.The zebrafish book:a guide for the laboratory use of zebrafish(Danio rerio)[M].5th ed.Eugene:University of Oregon Press,2007.
[3]杨冰洁.几种紫薇属植物染色体制片技术优化及原位杂交体系的建立[D].北京:北京林业大学,2013.Yang B J.Optimization of chromosome sectioning and establishment of FISH and GISH of Lagerstroemia L.[D].Beijing:Beijing Forestry University,2013(in Chinese).
[4]鲁翠云,张明昭,李超,等.荧光原位杂交技术在鱼类基因定位研究中的应用[J].水产学杂志,2013,26(4):51-57.Lu C Y,Zhang M Z,Li C,et al.Application of fluorescence in situ hybridization(FISH)techniques in gene location in fish[J].Chinese Journal of Fisheries,2013,26(4):51-57(in Chinese).
[5]Cao J M,Li S Q,Zhang H W,et al.High mobility group B proteins regulate mesoderm formation and dorsoventral patterning during zebrafish and Xenopus early development[J].Mechanisms of Development,2012,129(9-12):263-274.
[6]Lauter G,S?ll I,Hauptmann G.Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain[J].Neural Development,2011,6:10.
[7]陈琼霞,镇鸿燕,李艳,等.基于石蜡包埋组织荧光原位杂交的技术问题[J].中国医药指南,2013,11(13):382.Chen Q X,Zhen H Y,Li Y,et al.Based on fluorescence in situ hybridization of paraffin-embedded tissue[J].Guide of China Medicine,2013,11(13):382(in Chinese).
[8]靳雁斌,丁学锋,司慧远,等.小鼠Cpne5基因mRNA水平的表达分布及其在胚胎的表达定位[J].标记免疫分析与临床,2013,20(2):93-96.Jin Y B,Ding X F,Si H Y,et al.Expression of mouse Cpne5 gene mRNA in various tissues and its location in embryo[J].Labeled Immunoassays and Clinical Medicine,2013,20(2):93-96(in Chinese).
[9]蒋丽,王阳阳,程安达,等.鲤鱼(Cyprinus carpio)外异蛋白A受体Edar基因的克隆及表达定位[J].生物技术通报,2013(12):99-107.Jiang L,Wang Y Y,Cheng A D,et al.Preliminary study of Edar gene cloning and localization of expression in Cyprinus carpio[J].Biotechnology Bulletin,2013(12):99-107(in Chinese).
[10]叶寒青,陈松林,刘洋,等.表达绿色荧光蛋白基因的花鲈胚胎干细胞株的建立及其体外分化[J].高技术通讯,2006,16(1):61-66.Ye H Q,Chen S L,Liu Y,et al.Establishment and differentiation in vitro of sea perch embryonic stem cell line expressing GFP gene[J].Chinese High Technology Letters,2006,16(1):61-66(in Chinese).
[11]曹建萌,卢迈新,叶星,等.尼罗罗非鱼整胚原位杂交技术的建立和初步应用[J].水产学报,2014,38(11):1847-1854.Cao J M,Lu M X,Ye X,et al.A novel protocol of whole mount in situ hybridization(WISH)and its primary application in Nile tilapia[J].Journal of Fisheries of China,2014,38(11):1847-1854(in Chinese).
[12]杨永升,李宁,邓学梅,等.黑素皮质素受体1:哺乳动物黑色素形成中的关键基因[J].遗传,2004,26(4):544-550.Yang Y S,Li N,Deng X H,et al.MC1R:the key gene in mammalian melanin synthesis[J].Hereditas(Beijing),2004,26(4):544-550(in Chinese).
[13]FitzGerald L M,Fryer J L,Dwyer T,et al.Effect of MELANOTAN?,[Nle4,D-Phe7]-α-MSH,on melanin synthesis in humans with MC1R variant alleles[J].Peptides,2006,27(2):388-394.
[14]Adalsteinsson S,Hersteinsson P,Gunnarsson E.Fox colors in relation to colors in mice and sheep[J].Journal of Heredity,1987,78(4):235-237.
[15]孙静.红褐色突变貉毛色基因MC1R的序列分析及其表达水平的研究[D].青岛:青岛农业大学,2012.Sun J.Sequence analysis and expression level study on MC1R gene in reddish-brown raccoon dog[D].Qingdao:Qingdao Agricultural University,2012(in Chinese).
[16]任玉红,杨刚,范瑞文,等.黑素皮质素受体1(MC1R)在不同毛色羊驼皮肤组织中的表达与定位研究[J].畜牧兽医学报,2012,43(7):1049-1055.Ren Y H,Yang G,Fan R W,et al.Expression and immunolocalization of melanocortin receptor 1(MC1R)in alpaca skin with different coat color[J].Acta Veterinaria et Zootechnica Sinica,2012,43(7):1049-1055(in Chinese).
[17]胡建尊,李康乐,项松平,等.瓯江彩鲤体色调控相关因子MC1R的克隆与表达分析[J].上海海洋大学学报,2013,22(4):518-523.Hu J Z,Li K L,Xiang S P,et al.Cloning and expression analysis of color-related regulation factor MC1R in Oujiang color common carp[J].Journal of Shanghai Ocean University,2013,22(4):518-523(in Chinese).
[18]蒋燕玲.橘色双冠丽鱼体色发育变化及体色相关基因TYR的克隆与表达研究[D].上海:上海海洋大学,2016.Jiang Y L.Body color variation and cloning,expression analysis of TYR gene in Amphilophus citrinellus[D].Shanghai:Shanghai Ocean University,2016(in Chinese).
[19]孙桂金,潘杰,刘可春,等.苯硫脲对斑马鱼黑色素生成及早期发育的影响[J].水产科学,2011,30(7):387-390.Sun G J,Pan J,Liu K C,et al.Effects of N-phenylthiourea(PTU)on melanogenesis and early development in zebrafish(Danio rerio)[J].Fisheries Science,2011,30(7):387-390(in Chinese).
[20]徐永江,朱学武,柳学周,等.半滑舌鳎(Cynoglossus semilaevis)黑色素富集激素基因的克隆和表达[J].渔业科学进展,2017,38(1):81-90.Xu Y J,Zhu X B,Liu X Z,et al.Cloning and expression of melanin-concentrating hormone in half-smooth tongue sole(Cynoglossus semilaevis)[J].Progress in Fishery Sciences,2017,38(1):81-90(in Chinese).
[21]王东,方军,厉有名,等.链霉蛋白酶提高胃镜检查图像可见度的前瞻性、多中心、双盲、随机对照临床研究[J].中华消化内镜杂志,2013,30(11):604-607.Wang D,Fang J,Li Y M,et al.Effectiveness of premedication with pronase for visualization of the mucosa during endoscopy:a double-blinded,multicenter,prospective,randomized study[J].Chinese Journal of Digestive Endoscopy,2013,30(11):604-607(in Chinese).
[22]纪莉莎,管泱,姜昕.链霉蛋白酶在胃镜检查中的临床应用[J].中国继续医学教育,2015,7(23):129-130.Ji L S,Guan Y,Jiang X.Application of pronase in gastroscopy examination[J].China Continuing Medical Education,2015,7(23):129-130(in Chinese).
[23]樊竑冶,彭忠禄,谢华平,等.斑马鱼整体原位杂交的技术改良[J].激光生物学报,2010,19(1):115-119.Fan H Y,Peng Z L,Xie H P,et al.Improvement of whole-mount in situ hybridization to zebrafish embryos[J].Acta Laser Biology Sinica,2010,19(1):115-119(in Chinese).
[24]张春霞,刘峰.斑马鱼高分辨率整胚原位杂交实验方法与流程[J].遗传,2013,35(4):522-528.Zhang C X,Liu F.A brief protocol for high-resolution whole mount in situ hybridization in zebrafish[J].Hereditas(Beijing),2013,35(4):522-528(in Chinese).
[25]韦敏侠,宋红梅,祁宝伦,等.橘色双冠丽鱼胚后色素细胞发育与体色变化[J].上海海洋大学学报,2015,24(1):28-35.Wei M X,Song H M,Qi B L,et al.Pigment cells development and body color variation of postembryonic development in Amphilophus citrinellus(Günther1864)[J].Journal of Shanghai Ocean University,2015,24(1):28-35(in Chinese).
[26]程炜轩,许国焕,熊达,等.黄颡鱼黑色素细胞原代培养及迁移相关基因克隆分析[J].生态毒理学报,2014,9(6):1035-1040.Cheng W X,Xu G H,Xiong D,et al.Primary culturing of melanoma cells and cloning analysis of migrationrelated gene in Pelteobagrus fulvidraco[J].Asian Journal of Ecotoxicology,2014,9(6):1035-1040(in Chinese).
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