本文選題：黃河鯉　切入點(diǎn)：體色　出處：《河南師范大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：黃河鯉(Cyprinus carpio haematopterus),隸屬于鯉形目(Cypriniformes),鯉科(Cyprinidae),鯉屬(Cyprinus),因其金鱗赤尾,肉質(zhì)鮮美而聞名于世,同淞江鱸、興凱湖湆、松花江鱖被譽(yù)為我國(guó)四大淡水名魚(yú),是黃河流域主要的名貴優(yōu)質(zhì)魚(yú)類之一。目前對(duì)于魚(yú)類體色等方面的研究已經(jīng)開(kāi)展很多,主要集中于模式生物斑馬魚(yú)(Danio rerio)、青溕(Oryzias latipes)或者一些觀賞魚(yú)上,但對(duì)黃河鯉體色的研究還相對(duì)較少。Mitfa基因是神經(jīng)嵴細(xì)胞向黑色素細(xì)胞定向發(fā)育過(guò)程中最早的發(fā)生標(biāo)志之一,并且其通過(guò)調(diào)控酪氨酸酶基因家族的表達(dá)來(lái)參與到黑色素細(xì)胞的分化和發(fā)育。Tyr作為酪氨酸酶基因家族的成員之一,是黑色素合成過(guò)程中的關(guān)鍵限速酶,其表達(dá)和活性決定黑色素生成的速度和產(chǎn)量。本文以黃河鯉為實(shí)驗(yàn)材料,選取黑色素合成通路中的關(guān)鍵基因mitfa和tyr作為研究對(duì)象,克隆得到mitfa和tyr基因的全長(zhǎng),研究?jī)苫蛟邳S河鯉各組織中的表達(dá)分布,并觀察了黃河鯉體色的形成過(guò)程,檢測(cè)兩基因在體色形成各時(shí)期的表達(dá),進(jìn)一步探討mitfa和tyr基因在魚(yú)類體色形成中的作用。1、黃河鯉mitfa基因cDNA全長(zhǎng)的克隆和表達(dá)定位以黃河鯉黑色皮膚提取的總RNA作為模板,克隆得到mitfa基因cDNA全長(zhǎng)(No.KT984198),實(shí)時(shí)熒光定量分析其在8個(gè)組織中的表達(dá)情況,免疫組化方法檢測(cè)其在兩種不同顏色皮膚的表達(dá)及定位。結(jié)果顯示:黃河鯉mitfa基因cDNA全長(zhǎng)為1861 bp,包含1462 bp的開(kāi)放閱讀框,編碼486個(gè)氨基酸,與錦鯉的相似性為95.7%。熒光定量結(jié)果顯示mitfa在黃河鯉的8個(gè)組織中均有表達(dá),并且在肌肉中的表達(dá)顯著高于其它組織(P0.05)。免疫組化結(jié)果顯示:Mitfa在黑色皮膚和銀色皮膚中均有表達(dá),并且在黑色皮膚中的表達(dá)比在銀色皮膚中表達(dá)強(qiáng)烈。2、黃河鯉tyr基因cDNA全長(zhǎng)的克隆和表達(dá)定位以黃河鯉黑色皮膚總RNA為模板,RACE方法克隆tyr基因cDNA全長(zhǎng),實(shí)時(shí)熒光定量分析在11個(gè)組織中的表達(dá)情況,免疫組化方法檢測(cè)兩種不同顏色皮膚中的表達(dá)及定位。結(jié)果顯示:黃河鯉Try基因cDNA全長(zhǎng)1717 bp(No.KY305667),其中ORF長(zhǎng)1605 bp,編碼535個(gè)氨基酸,5'-UTR區(qū)41 bp,3'-UTR區(qū)71 bp。蛋白同源分析發(fā)現(xiàn)黃河鯉Tyr與鯉科魚(yú)類相似性最高,與哺乳類及鳥(niǎo)類等脊椎動(dòng)物相似性最低。系統(tǒng)進(jìn)化分析顯示黃河鯉Tyr與錦鯉和斑馬魚(yú)等鯉科魚(yú)類的親緣關(guān)系最近。熒光定量結(jié)果發(fā)現(xiàn)tyr在黃河鯉不同組織中均有表達(dá),肌肉中的表達(dá)量最高,之后是黑色皮膚,在銀色皮膚中的表達(dá)量最低。免疫組織化學(xué)結(jié)果顯示Tyr在黑色皮膚的黑色素細(xì)胞和粘液細(xì)胞中有表達(dá),在銀色皮膚沒(méi)有表達(dá)。3、mitfa和tyr基因在黃河鯉體色發(fā)生中的表達(dá)分析觀察描述了黃河鯉體色的發(fā)生過(guò)程,并對(duì)黃河鯉成魚(yú)背部鱗片、腹部鱗片及鰭條的色素細(xì)胞的組成進(jìn)行了觀察。利用實(shí)時(shí)熒光定量PCR分析mitfa和tyr基因在黃河鯉體色形成過(guò)程中的表達(dá)情況。觀察結(jié)果顯示黃河鯉具有虹彩細(xì)胞、黃色素細(xì)胞和黑色素細(xì)胞三種色素細(xì)胞。對(duì)黃河鯉體色發(fā)生的觀察顯示,1 dph(1 days past hatch)在黃河鯉背部和腹部均有黑色素細(xì)胞;2 dph時(shí)黃色素細(xì)胞出現(xiàn),黑色素細(xì)胞數(shù)量增多;之后黑色素細(xì)胞和黃色素細(xì)胞數(shù)量逐漸增多直至遍布全身;31 dph~41 dph,虹彩色素細(xì)胞和鱗片開(kāi)始出現(xiàn)。51 dph時(shí)體色基本形成。熒光定量結(jié)果顯示:mitfa和tyr基因在黃河鯉體色形成各階段均有表達(dá),并且在51 dph達(dá)到最高。其中mitfa和tyr基因在黃河鯉體色形成過(guò)程中的表達(dá)趨勢(shì)基本相同即隨著黑色素細(xì)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.

【學(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

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