黄河鲤体色发生观察及mitfa和tyr基因cDNA的克隆与组织表达研究

发布时间：2018-03-17 06:12

本文选题：黄河鲤　切入点：体色　出处：《河南师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：黄河鲤(Cyprinus carpio haematopterus),隶属于鲤形目(Cypriniformes),鲤科(Cyprinidae),鲤属(Cyprinus),因其金鳞赤尾,肉质鲜美而闻名于世,同淞江鲈、兴凯湖湆、松花江鳜被誉为我国四大淡水名鱼,是黄河流域主要的名贵优质鱼类之一。目前对于鱼类体色等方面的研究已经开展很多,主要集中于模式生物斑马鱼(Danio rerio)、青溕(Oryzias latipes)或者一些观赏鱼上,但对黄河鲤体色的研究还相对较少。Mitfa基因是神经嵴细胞向黑色素细胞定向发育过程中最早的发生标志之一,并且其通过调控酪氨酸酶基因家族的表达来参与到黑色素细胞的分化和发育。Tyr作为酪氨酸酶基因家族的成员之一,是黑色素合成过程中的关键限速酶,其表达和活性决定黑色素生成的速度和产量。本文以黄河鲤为实验材料,选取黑色素合成通路中的关键基因mitfa和tyr作为研究对象,克隆得到mitfa和tyr基因的全长,研究两基因在黄河鲤各组织中的表达分布,并观察了黄河鲤体色的形成过程,检测两基因在体色形成各时期的表达,进一步探讨mitfa和tyr基因在鱼类体色形成中的作用。1、黄河鲤mitfa基因cDNA全长的克隆和表达定位以黄河鲤黑色皮肤提取的总RNA作为模板,克隆得到mitfa基因cDNA全长(No.KT984198),实时荧光定量分析其在8个组织中的表达情况,免疫组化方法检测其在两种不同颜色皮肤的表达及定位。结果显示:黄河鲤mitfa基因cDNA全长为1861 bp,包含1462 bp的开放阅读框,编码486个氨基酸,与锦鲤的相似性为95.7%。荧光定量结果显示mitfa在黄河鲤的8个组织中均有表达,并且在肌肉中的表达显著高于其它组织(P0.05)。免疫组化结果显示:Mitfa在黑色皮肤和银色皮肤中均有表达,并且在黑色皮肤中的表达比在银色皮肤中表达强烈。2、黄河鲤tyr基因cDNA全长的克隆和表达定位以黄河鲤黑色皮肤总RNA为模板,RACE方法克隆tyr基因cDNA全长,实时荧光定量分析在11个组织中的表达情况,免疫组化方法检测两种不同颜色皮肤中的表达及定位。结果显示:黄河鲤Try基因cDNA全长1717 bp(No.KY305667),其中ORF长1605 bp,编码535个氨基酸,5'-UTR区41 bp,3'-UTR区71 bp。蛋白同源分析发现黄河鲤Tyr与鲤科鱼类相似性最高,与哺乳类及鸟类等脊椎动物相似性最低。系统进化分析显示黄河鲤Tyr与锦鲤和斑马鱼等鲤科鱼类的亲缘关系最近。荧光定量结果发现tyr在黄河鲤不同组织中均有表达,肌肉中的表达量最高,之后是黑色皮肤,在银色皮肤中的表达量最低。免疫组织化学结果显示Tyr在黑色皮肤的黑色素细胞和粘液细胞中有表达,在银色皮肤没有表达。3、mitfa和tyr基因在黄河鲤体色发生中的表达分析观察描述了黄河鲤体色的发生过程,并对黄河鲤成鱼背部鳞片、腹部鳞片及鳍条的色素细胞的组成进行了观察。利用实时荧光定量PCR分析mitfa和tyr基因在黄河鲤体色形成过程中的表达情况。观察结果显示黄河鲤具有虹彩细胞、黄色素细胞和黑色素细胞三种色素细胞。对黄河鲤体色发生的观察显示,1 dph(1 days past hatch)在黄河鲤背部和腹部均有黑色素细胞;2 dph时黄色素细胞出现,黑色素细胞数量增多;之后黑色素细胞和黄色素细胞数量逐渐增多直至遍布全身;31 dph~41 dph,虹彩色素细胞和鳞片开始出现。51 dph时体色基本形成。荧光定量结果显示:mitfa和tyr基因在黄河鲤体色形成各阶段均有表达,并且在51 dph达到最高。其中mitfa和tyr基因在黄河鲤体色形成过程中的表达趋势基本相同即随着黑色素细胞的增加而增加。
[Abstract]:The Yellow River carp (Cyprinus carpio haematopterus), belongs to Cypriniformes, Cyprinidae (Cypriniformes) (Cyprinidae), Cyprinidae (Cyprinus), because of its Jinlin Akao, delicious and famous, with the Songjiang perch, Qi Xingkai Lake, Songhua River is known as the Mandarin of China's four major freshwater fishes the Yellow River, is one of the major watersheds of the precious high quality fish. The present study for fish body color and other aspects have been carried out many, mainly concentrated in the zebrafish (Danio rerio), green Meng (Oryzias latipes) or some fish, but the research on the Yellow River carp body color is relatively less.Mitfa gene is one of the earliest signs the process of neural crest cell development targeted melanoma cells, and its expression through regulation of tyrosinase gene family to participate in the development and differentiation of.Tyr melanoma cells as a member of the tyrosinase gene family, is the synthesis of melanin The key enzyme in the process of its expression and activity determines the melanin production rate and yield. This paper takes the Yellow River carp as experimental materials, selection of melanin synthesis pathway of key genes mitfa and Tyr as the research object, the cloned full-length mitfa and Tyr genes, two studies for expression in various tissues of carp in the Yellow River the distribution, and to observe the forming process of the Yellow River carp body color, two gene expression detection of each period in the body, to further explore the role of.1 mitfa and TYR gene in fish body formation, cloning of the Yellow River carp mitfa gene full-length cDNA and total RNA as a template for positioning in the Yellow River carp black skin extract the expression of cloned full-length mitfa cDNA (No.KT984198) gene and its expression in 8 tissues of real-time fluorescent quantitative PCR, immunohistochemistry detection in two different colors of skin. The results showed that the expression and localization of: The Yellow River carp mitfa gene cDNA was 1861 BP, containing an open reading frame of 1462 BP encoding 486 amino acids, and the similarity of carp display mitfa in 8 organizations of the Yellow River carp were expressed as 95.7%. fluorescence quantitative results, and the expression in muscle was significantly higher than that in other tissues (P0.05). The results of immunohistochemistry display: Mitfa in black skin and silver skin tissues, and the expression in the black skin than strongly expressed.2 in silver skin, cloning the Yellow River carp TYR gene full-length cDNA expression and localization in the Yellow River black carp skin total RNA as template, the RACE method to clone TYR gene full-length cDNA expression in 11 groups in the analysis of real time fluorescent quantitative immunohistochemical method to detect the expression and localization of two different colors in the skin. The results showed: the Yellow River carp full-length Try gene cDNA 1717 BP (No.KY305667), ORF 1605 BP, encoding 535 Amino acids, 5'-UTR 41 BP, 3'-UTR 71 bp. homology analysis showed that Tyr and the Yellow River carp Cyprinidae highest similarity with mammals and birds and other vertebrates, the lowest similarity. Phylogenetic analysis showed genetic relationship between the Yellow River carp and Koi Carp and zebrafish Tyr and Cyprinids. Recent results in fluorescence quantitative Tyr the Yellow River carp was expressed in different tissues, the expression of the muscles in the highest, after the skin is black, silver in the skin in the lowest expression. Immunohistochemical staining showed that Tyr expression in melanoma cells and mucous cells with dark skin, no expression of.3 in silver skin, describes the process of observation and analysis of the Yellow River carp body color expression of mitfa and TYR gene in the Yellow River carp in color, and the the Yellow River carp fish scales back, abdominal scales and fin composed of pigment cells were observed by fluorescence. Quantitative PCR analysis of mitfa and TYR gene expression in the process of the formation of the Yellow River carp. Color observation results show that the Yellow River carp with iridescent yellow pigment cells, cells and melanoma cells of three kinds of pigment cells. According to observation of the Yellow River carp body occurred, 1 DPH (1 days past hatch) in the Yellow River carp back and abdomen were black pigment cell; yellow pigment cells appeared at 2 DPH, the number of melanoma cells increased; after the number of melanoma cells and pigment cells gradually increased until all over the body; 31 dph~41 DPH, iris pigment cells and.51 DPH color scales began to appear basically formed. Quantitative results showed that mitfa and Tyr genes in each stage were formed the Yellow River carp body color expression, and reached the highest at 51 DPH. The mitfa and Tyr genes in the Yellow River carp expression trend in the process of color formation is basically the same with the increase of melanoma cells increased.

【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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