本文選題：美洲水貂 + 黑色素��； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文

【摘要】：水貂(Neovison vison),是一種珍貴毛皮動(dòng)物,其毛皮主要用于制裘,因此,被毛顏色是決定貂皮質(zhì)量與價(jià)值的一種重要經(jīng)濟(jì)性狀,深入解析水貂被毛色素沉積機(jī)理在新型毛色品種培育過(guò)程中具有重要的理論與實(shí)踐意義。為了更好地明確水貂被毛顏色表型形成的遺傳調(diào)控機(jī)制,本研究以具有顯著毛色差異的金州黑水貂(黑色)、吉林白水貂(白色)、銀藍(lán)水貂(灰色)、咖啡水貂(棕褐色)和珍珠水貂(珍珠色)為研究對(duì)象,利用組織學(xué)、細(xì)胞學(xué)、分子生物學(xué)及高通量RNA-seq轉(zhuǎn)錄組測(cè)序技術(shù),系統(tǒng)開(kāi)展了水貂被毛黑色素含量差異分析、成熟黑色素細(xì)胞組織學(xué)定位檢測(cè)、毛色關(guān)聯(lián)基因克隆及其遺傳變異位點(diǎn)篩查、毛色基因mRNA差異表達(dá)分析與基于高通量RNA-seq皮膚轉(zhuǎn)錄組挖掘色素沉積相關(guān)基因等六個(gè)相互關(guān)聯(lián)的試驗(yàn)研究,主要研究?jī)?nèi)容及獲得的研究結(jié)果如下:1.通過(guò)測(cè)定換毛期和毛皮成熟期金州黑水貂、銀藍(lán)水貂和吉林白水貂被毛黑色素含量,比較3種不同被毛顏色水貂毛纖維中總黑色素(Total melanin,TM)、真黑色素(Eumelanin,EM)及褐黑色素(Pheomelanin,PM)含量的差異,同時(shí)分析不同時(shí)期被毛黑色素含量的變化。結(jié)果表明,換毛期和毛皮成熟期,黑色水貂被毛TM、EM和PM含量均最高,隨著被毛顏色變淺,3種黑色素含量均呈降低趨勢(shì)。單因素方差分析顯示,黑色水貂中,毛皮成熟期被毛TM和PM含量分別是換毛期的1.17和1.20倍(P0.01),EM含量顯著高于換毛期(P0.05);白色水貂毛皮成熟期被毛TM和PM含量分別是換毛期的1.27和1.22倍(P0.05),EM含量略高于換毛期,差異不顯著(P0.05);銀藍(lán)水貂被毛TM、EM和PM含量在毛皮成熟期和換毛期差異不顯著(P0.05)。PM含量決定了水貂灰色和白色被毛表型的發(fā)生,EM含量可能與黑色被毛表型存在關(guān)聯(lián),在水貂換毛過(guò)程中,存在于皮膚黑色素小體中的黑色素由毛囊定向轉(zhuǎn)移到毛干。2.利用甲苯胺藍(lán)染色、多巴氧化酶法(dopa)及dopa-甲苯胺藍(lán)聯(lián)合染色三種方法,分析黑色、灰色和白色被毛水貂皮膚組織中成熟黑色素細(xì)胞的分布特性。甲苯胺藍(lán)染色結(jié)果表明,3種毛色水貂皮膚、毛囊及其附屬物結(jié)構(gòu)特征比較明顯,其中有核細(xì)胞的細(xì)胞核被染成藍(lán)色,毛皮成熟期水貂表皮相對(duì)真皮較薄,毛根下端形成杵狀毛(club hair),未見(jiàn)明顯凹陷的毛乳頭,推斷水貂毛囊處于退行期(catagen)后期。dopa染色與dopa-甲苯胺藍(lán)復(fù)染鏡檢結(jié)果均顯示,黑色水貂表皮多巴陽(yáng)性著色較灰色水貂深,且黑色素顆粒呈連續(xù)分布,灰色水貂表皮陽(yáng)性著色區(qū)域顏色較淺,呈間斷分布。多巴陽(yáng)性呈“團(tuán)狀”大量聚集于黑色被毛水貂皮膚毛囊頂端,毛囊外根鞘和毛根的髓質(zhì)中有少量著色帶,沿毛干方向,顏色逐漸變淡;灰色被毛毛囊頂端多巴陽(yáng)性著色帶較少,呈“稀疏顆�！狈植�,毛囊外根鞘陽(yáng)性著色較黑色被毛弱。白色被毛水貂皮膚組織中未見(jiàn)明顯多巴陽(yáng)性著色區(qū)域。毛囊頂端的成熟黑色素細(xì)胞是水貂被毛色素沉積的主要細(xì)胞學(xué)基礎(chǔ),該部位成熟黑色素細(xì)胞數(shù)量及酪氨酸酶活性是決定其毛色差異的直接因素,表皮中的黑色素細(xì)胞可能并不參與水貂被毛色素沉積。3.采用PCR、克隆測(cè)序技術(shù)獲得水貂MC1R、TYR和Agouti基因核苷酸序列,通過(guò)比較基因組學(xué)方法對(duì)3個(gè)基因進(jìn)行了鑒定及生物信息學(xué)分析。結(jié)果表明,美洲水貂MC1R基因長(zhǎng)度為1324 bp(GenBank登錄號(hào):KJ152766),編碼序列長(zhǎng)954 bp,編碼317個(gè)氨基酸,MC1R蛋白多肽鏈存在1個(gè)低復(fù)雜度結(jié)構(gòu)域和7個(gè)典型跨膜區(qū),具有細(xì)胞膜受體典型結(jié)構(gòu)。水貂TYR基因(Gen Bank登錄號(hào):KJ716783)5個(gè)完整的外顯子長(zhǎng)度分別為:819 bp(外顯子1)、217 bp(外顯子2)、148 bp(外顯子3)、182 bp(外顯子4)和230 bp(外顯子5),編碼區(qū)全長(zhǎng)1596 bp,編碼531個(gè)氨基酸,由18個(gè)氨基酸的信號(hào)肽和513個(gè)氨基酸的成熟肽組成。水貂Agouti基因長(zhǎng)度為4231 bp,包括部分內(nèi)含子1(424 bp)、完整外顯子2(160 bp)、外顯子3(62 bp)、內(nèi)含子2(1256 bp)和部分內(nèi)含子3(2329 bp)。4.采集金州黑水貂、銀藍(lán)水貂、吉林白水貂、咖啡水貂和珍珠色水貂共計(jì)430份血液,利用PCR產(chǎn)物直接測(cè)序技術(shù)對(duì)MC1R、TYR和Agouti基因編碼區(qū)及非編碼區(qū)開(kāi)展SNPs檢測(cè),將不同基因突變位點(diǎn)形成的基因型與水貂毛色表型進(jìn)行了關(guān)聯(lián)分析。結(jié)果表明,水貂MC1R基因編碼區(qū)不存在多態(tài)性,編碼區(qū)上游存在缺失突變位點(diǎn):g.132_135delAGTG,銀藍(lán)水貂在該位點(diǎn)的多態(tài)信息含量為0.3268,屬中度多態(tài),暗示該突變位點(diǎn)可能影響銀藍(lán)水貂的被毛顏色,或者與調(diào)控銀藍(lán)水貂灰色被毛表型的主效基因連鎖。TYR基因外顯子4和5不具有多態(tài)性,外顯子1無(wú)義SNPs(g.138TA)與吉林白水貂白色被毛表型極顯著相關(guān)(P0.0001)。Agouti基因內(nèi)含子2中g(shù).1189CT位點(diǎn)的CT基因型與吉林白水貂白色被毛表型存在關(guān)聯(lián);內(nèi)含子3上g.252CT位點(diǎn)CT基因型與珍珠水貂的毛色存在關(guān)聯(lián),5個(gè)SNPs(g.290AC、g.298GC、g.340AG、g.343TC和g.379TC)與2個(gè)缺失突變位點(diǎn)(g.472_473delCA和g.974_977delCTCT)在不同毛色水貂群體內(nèi)分別處于緊密連鎖狀態(tài)且可能與水貂淺色被毛表型相關(guān)。5.采用qRT-PCR技術(shù)分析了金州黑水貂、銀藍(lán)水貂和吉林白水貂毛皮成熟期背部皮膚組織MC1R、TYR和Agouti基因mRNA表達(dá)量。結(jié)果表明,毛皮成熟期,3個(gè)基因在3種毛色水貂皮膚中均有表達(dá)。在MC1R基因mRNA表達(dá)上,白色與黑色、灰色差異極顯著(P0.01),黑色與灰色差異不顯著(P0.05),相對(duì)表達(dá)量順序?yàn)?白色灰色黑色。黑色、灰色被毛水貂TYR基因mRNA表達(dá)量分別是白色被毛水貂的3.2535和1.8933倍,黑色與灰色、白色被毛水貂差異極顯著(P0.01),相對(duì)表達(dá)量順序?yàn)?黑色灰色白色。黑色、灰色被毛水貂Agouti基因mRNA表達(dá)量分別是白色被毛水貂的1.2515和0.9501倍,但三者間差異不顯著(P0.05),相對(duì)表達(dá)量順序?yàn)?黑色白色灰色。6.基于高通量RNA-seq技術(shù)測(cè)定了金州黑水貂、銀藍(lán)水貂和吉林白水貂皮膚組織轉(zhuǎn)錄組,利用qRT-PCR技術(shù)對(duì)可能影響水貂被毛色素沉積的關(guān)鍵基因進(jìn)行了mRNA定量表達(dá)驗(yàn)證。結(jié)果表明,獲得毛皮成熟期水貂皮膚轉(zhuǎn)錄組原始數(shù)據(jù)約33.19 G,403,725個(gè)Unigene成功注釋到7個(gè)生物信息學(xué)數(shù)據(jù)庫(kù)中。BLM_S vs WHM_S(黑色vs白色)、BLM_S vs SBM_S(黑色vs灰色)和WHM_S vs SBM_S(白色vs灰色)組合中分別存在12,557、4,334和10,643個(gè)差異表達(dá)基因,3個(gè)組合共有的差異表達(dá)基因?yàn)?,735個(gè)。3個(gè)組合中分別存在20、10和17個(gè)參與水貂被毛色素沉積的差異表達(dá)基因,KITLG、LEF1、DCT、TYRP1、PMEL、TYR、Myo5a、Rab27a和SLC7A11基因可能從黑色素細(xì)胞發(fā)育、黑素小體成分及其前體、黑素小體轉(zhuǎn)運(yùn)和真黑色素與褐黑色素合成轉(zhuǎn)變開(kāi)關(guān)這4個(gè)生物學(xué)過(guò)程調(diào)控水貂毛色表型。
[Abstract]:Mink (Neovison vison) is a kind of precious fur animal, whose fur is mainly used in making fur. Therefore, the coat color is an important economic character that determines the quality and value of mink skin. It is of great theoretical and practical significance to deeply analyze the mechanism of feather pigment deposition in mink in the process of breeding new hair color varieties. The genetic regulation mechanism formed by the color phenotypes of the hair is studied in Jinzhou Black Mink (black), Jilin mink (white), Silver Blue mink (gray), coffee mink (brown) and Pearl mink (pearl color), using histological, cytology, molecular biology and high throughput RNA-seq transcriptome sequencing techniques. The system carried out six interrelated studies on the difference analysis of mink's melanin content, histology localization detection of mature melanocytes, hair color association gene cloning and genetic mutation site screening, differential expression analysis of hair color gene mRNA and mining of pigment sedimentation related genes based on high throughput RNA-seq skin transcriptional group. The main research content and the results obtained are as follows: 1. by measuring the content of hair melanin in Jinzhou mink, Silver Blue mink and Jilin mink, total melanin (Total melanin, TM), true melanin (Eumelanin, EM) and Pheomelanin, PM content in mink wool fibers were compared. The results showed that the contents of TM, EM and PM in the Black Mink were both the highest, and the 3 kinds of melanin content decreased with the shallower color, and the single factor variance analysis showed that in the Black Mink, the fur matured period was TM and PM respectively. The content of EM was 1.17 and 1.20 times (P0.01), and the content of TM and PM in the mature period of white mink was 1.27 and 1.22 times (P0.05) respectively, and the content of EM was slightly higher than that of the hairy period (P0.05), and the difference of TM, EM and PM content of silvery blue mink was not significant (P0.05). The content of.PM determines the occurrence of gray and white coat type of mink. The content of EM may be associated with the black coat type. In the process of the mink, the melanin in the skin melanin is transferred from the hair follicle to the dry.2., using toluidine blue, DOPA and dopa- toluidine blue. Methods, the distribution characteristics of the mature melanocytes in the skin tissues of black, gray and white mink were analyzed. The results of toluidine blue staining showed that the structure characteristics of the skin, hair follicles and their appendages of 3 kinds of mink were more obvious, and the nuclei of the nuclear cells were dyed blue, and the skin of mink was thinner than the skin in the mature period. The lower end formed a pestle (club hair) and no obvious depression of the hair nipple. It was concluded that the mink hair follicle was in the degenerative period (catagen) and the.Dopa staining and the dopa- toluidine blue staining microscopy showed that the positive coloring of the Black Mink's epidermis was deeper than the gray mink, and the melanin granules were continuously distributed and the gray mink skin positive colored region pigments were found. The color is shallow and discontinuous. The positive "regiment" of DOPA is concentrated in the top of the skin hair follicle of the black mink. The medulla of the outer root sheath and the root of the hair follicle has a small number of color bands, and the color gradually dimmed along the direction of the hair. The Gray was less positive in the Mao Maonang's top DOPA and showed a "sparse particle" distribution and the outer root sheath of the hair follicle was positive. The color is weaker than black. There is no obvious DOPA positive staining area in the skin tissue of the white mink. Mature melanocytes at the top of the hair follicle are the main cytological basis for the deposition of mink's hair pigment. The number of mature melanocytes and tyrosinase activity in this part are the direct factors determining the difference in hair color, and the melanin in the epidermis The cells may not participate in the use of PCR for the mink's hair pigment deposition.3., and cloned sequencing technology to obtain the nucleotide sequences of mink MC1R, TYR and Agouti genes. The 3 genes were identified by comparative genomics and bioinformatics analysis. The results showed that the length of the mink MC1R base was 1324 BP (GenBank login number: KJ152766), and the coding was encoded. The sequence length is 954 BP, encoding 317 amino acids, MC1R protein polypeptide chain has 1 low complexity domains and 7 typical transmembrane regions, with the typical structure of cell membrane receptor. The length of 5 complete exons of mink TYR gene (Gen Bank login No. KJ716783) is 819 BP (exon 1), 217 BP (exon 2), 148 BP (exon 3), 182 BP (exon). 4) and 230 BP (exon 5), the whole length of the coding region is 1596 BP, encoding 531 amino acids, consisting of 18 amino acid signal peptides and 513 amino acid mature peptides. The length of the mink Agouti gene is 4231 BP, including partial intron 1 (424 BP), complete exon 2 (160), exon BP and partial inclusions (BP).4.. A total of 430 blood samples were collected from Jinzhou Black Mink, Silver Blue mink, Jilin White Mink, coffee mink, mink and pearl color mink. Using direct sequencing of PCR products, MC1R, TYR and Agouti gene coding regions and non coding regions were detected by SNPs, and the genotype of different gene mutation sites was associated with the hair color phenotype of mink. The results showed that There is no polymorphism in the coding region of the mink MC1R gene, and there is a missing mutation site in the upstream of the coding region: g.132_135delAGTG, the polymorphism information content of the silver blue mink is 0.3268, which is moderately polymorphic, suggesting that the mutation site may affect the color of the silvery mink's wool, or it is linked to the main effect gene regulating the gray blue mink of the silvery blue mink. The exon 4 and 5 of the.TYR gene were not polymorphic. The exon 1 nonsense SNPs (g.138TA) was significantly related to the white coat type of the Ji Linbai mink (P0.0001), the CT genotype of the g.1189CT locus in the intron 2 of the.Agouti gene was associated with the White Mink white coat type of Jilin mink, and the CT genotype at the upper g.252CT site of the intron 3 and the hair color of the Pearl mink. In association, 5 SNPs (g.290AC, g.298GC, g.340AG, g.343TC and g.379TC) and 2 missing mutation sites (g.472_473delCA and g.974_977delCTCT) are closely linked in different hair color mink populations and may be associated with mink's light color surface type.5. using qRT-PCR techniques for the analysis of the Jinzhou mink, the silver blue mink and the Jilin white water. The expression of MC1R, TYR and Agouti gene mRNA expressed in the skin tissue of the back skin of mink skin. The results showed that the 3 genes were expressed in the skin of 3 kinds of hair color mink. In the MC1R gene mRNA expression, white and black, gray difference was very significant (P0.01), black and gray color difference was not significant (P0.05), and the relative expression order was white ash. The expression of TYR gene mRNA in black and gray was 3.2535 and 1.8933 times of the White Mink, black and gray, and white in mink (P0.01). The order of relative expression was black gray white, black, and grey wool mink Agouti gene mRNA expression was 1.2515 and 0.9 of White Mink, respectively. 501 times, but the differences among the three were not significant (P0.05). The order of relative expression was: black white gray.6. was based on high throughput RNA-seq technique to determine the skin tissue transcriptional group of Jinzhou mink, Silver Blue mink and Jilin mink, and qRT-PCR technique was used to verify the mRNA quantitative expression of the key genes that may affect the coat pigments of mink. The results showed that the original data of the skin transcriptional group of mink skin were about 33.19 G, and 403725 Unigene were successfully annotated to.BLM_S vs WHM_S (black vs white) in 7 bioinformatics databases, BLM_S vs SBM_S (black vs grey) and WHM_S vs, and 10643 differentially expressed genes in the combination of the BLM_S vs SBM_S (black vs grey) and WHM_S vs. 20,10 and 17 differentially expressed genes, KITLG, LEF1, DCT, TYRP1, PMEL, TYR, Myo5a, Rab27a, and SLC7A11, may be developed from melanocytes, melanosomes and precursors, melanosomes and real melanin and browning in the 3 combinations of the 1735.3 combinations. The 4 biological processes of melanin synthesis switch regulate the phenotype of mink hair color.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S865.22
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本文編號(hào)：2014686