水貂被毛色素沉积机理及基于高通量RNA-seq皮肤转录组注释研究

发布时间：2018-06-13 16:45

  本文选题：美洲水貂 + 黑色素　； 参考：《中国农业科学院》2016年博士论文

【摘要】：水貂(Neovison vison),是一种珍贵毛皮动物,其毛皮主要用于制裘,因此,被毛颜色是决定貂皮质量与价值的一种重要经济性状,深入解析水貂被毛色素沉积机理在新型毛色品种培育过程中具有重要的理论与实践意义。为了更好地明确水貂被毛颜色表型形成的遗传调控机制,本研究以具有显著毛色差异的金州黑水貂(黑色)、吉林白水貂(白色)、银蓝水貂(灰色)、咖啡水貂(棕褐色)和珍珠水貂(珍珠色)为研究对象,利用组织学、细胞学、分子生物学及高通量RNA-seq转录组测序技术,系统开展了水貂被毛黑色素含量差异分析、成熟黑色素细胞组织学定位检测、毛色关联基因克隆及其遗传变异位点筛查、毛色基因mRNA差异表达分析与基于高通量RNA-seq皮肤转录组挖掘色素沉积相关基因等六个相互关联的试验研究,主要研究内容及获得的研究结果如下:1.通过测定换毛期和毛皮成熟期金州黑水貂、银蓝水貂和吉林白水貂被毛黑色素含量,比较3种不同被毛颜色水貂毛纤维中总黑色素(Total melanin,TM)、真黑色素(Eumelanin,EM)及褐黑色素(Pheomelanin,PM)含量的差异,同时分析不同时期被毛黑色素含量的变化。结果表明,换毛期和毛皮成熟期,黑色水貂被毛TM、EM和PM含量均最高,随着被毛颜色变浅,3种黑色素含量均呈降低趋势。单因素方差分析显示,黑色水貂中,毛皮成熟期被毛TM和PM含量分别是换毛期的1.17和1.20倍(P0.01),EM含量显著高于换毛期(P0.05);白色水貂毛皮成熟期被毛TM和PM含量分别是换毛期的1.27和1.22倍(P0.05),EM含量略高于换毛期,差异不显著(P0.05);银蓝水貂被毛TM、EM和PM含量在毛皮成熟期和换毛期差异不显著(P0.05)。PM含量决定了水貂灰色和白色被毛表型的发生,EM含量可能与黑色被毛表型存在关联,在水貂换毛过程中,存在于皮肤黑色素小体中的黑色素由毛囊定向转移到毛干。2.利用甲苯胺蓝染色、多巴氧化酶法(dopa)及dopa-甲苯胺蓝联合染色三种方法,分析黑色、灰色和白色被毛水貂皮肤组织中成熟黑色素细胞的分布特性。甲苯胺蓝染色结果表明,3种毛色水貂皮肤、毛囊及其附属物结构特征比较明显,其中有核细胞的细胞核被染成蓝色,毛皮成熟期水貂表皮相对真皮较薄,毛根下端形成杵状毛(club hair),未见明显凹陷的毛乳头,推断水貂毛囊处于退行期(catagen)后期。dopa染色与dopa-甲苯胺蓝复染镜检结果均显示,黑色水貂表皮多巴阳性着色较灰色水貂深,且黑色素颗粒呈连续分布,灰色水貂表皮阳性着色区域颜色较浅,呈间断分布。多巴阳性呈“团状”大量聚集于黑色被毛水貂皮肤毛囊顶端,毛囊外根鞘和毛根的髓质中有少量着色带,沿毛干方向,颜色逐渐变淡;灰色被毛毛囊顶端多巴阳性着色带较少,呈“稀疏颗粒”分布,毛囊外根鞘阳性着色较黑色被毛弱。白色被毛水貂皮肤组织中未见明显多巴阳性着色区域。毛囊顶端的成熟黑色素细胞是水貂被毛色素沉积的主要细胞学基础,该部位成熟黑色素细胞数量及酪氨酸酶活性是决定其毛色差异的直接因素,表皮中的黑色素细胞可能并不参与水貂被毛色素沉积。3.采用PCR、克隆测序技术获得水貂MC1R、TYR和Agouti基因核苷酸序列,通过比较基因组学方法对3个基因进行了鉴定及生物信息学分析。结果表明,美洲水貂MC1R基因长度为1324 bp(GenBank登录号:KJ152766),编码序列长954 bp,编码317个氨基酸,MC1R蛋白多肽链存在1个低复杂度结构域和7个典型跨膜区,具有细胞膜受体典型结构。水貂TYR基因(Gen Bank登录号:KJ716783)5个完整的外显子长度分别为:819 bp(外显子1)、217 bp(外显子2)、148 bp(外显子3)、182 bp(外显子4)和230 bp(外显子5),编码区全长1596 bp,编码531个氨基酸,由18个氨基酸的信号肽和513个氨基酸的成熟肽组成。水貂Agouti基因长度为4231 bp,包括部分内含子1(424 bp)、完整外显子2(160 bp)、外显子3(62 bp)、内含子2(1256 bp)和部分内含子3(2329 bp)。4.采集金州黑水貂、银蓝水貂、吉林白水貂、咖啡水貂和珍珠色水貂共计430份血液,利用PCR产物直接测序技术对MC1R、TYR和Agouti基因编码区及非编码区开展SNPs检测,将不同基因突变位点形成的基因型与水貂毛色表型进行了关联分析。结果表明,水貂MC1R基因编码区不存在多态性,编码区上游存在缺失突变位点:g.132_135delAGTG,银蓝水貂在该位点的多态信息含量为0.3268,属中度多态,暗示该突变位点可能影响银蓝水貂的被毛颜色,或者与调控银蓝水貂灰色被毛表型的主效基因连锁。TYR基因外显子4和5不具有多态性,外显子1无义SNPs(g.138TA)与吉林白水貂白色被毛表型极显著相关(P0.0001)。Agouti基因内含子2中g.1189CT位点的CT基因型与吉林白水貂白色被毛表型存在关联;内含子3上g.252CT位点CT基因型与珍珠水貂的毛色存在关联,5个SNPs(g.290AC、g.298GC、g.340AG、g.343TC和g.379TC)与2个缺失突变位点(g.472_473delCA和g.974_977delCTCT)在不同毛色水貂群体内分别处于紧密连锁状态且可能与水貂浅色被毛表型相关。5.采用qRT-PCR技术分析了金州黑水貂、银蓝水貂和吉林白水貂毛皮成熟期背部皮肤组织MC1R、TYR和Agouti基因mRNA表达量。结果表明,毛皮成熟期,3个基因在3种毛色水貂皮肤中均有表达。在MC1R基因mRNA表达上,白色与黑色、灰色差异极显著(P0.01),黑色与灰色差异不显著(P0.05),相对表达量顺序为:白色灰色黑色。黑色、灰色被毛水貂TYR基因mRNA表达量分别是白色被毛水貂的3.2535和1.8933倍,黑色与灰色、白色被毛水貂差异极显著(P0.01),相对表达量顺序为:黑色灰色白色。黑色、灰色被毛水貂Agouti基因mRNA表达量分别是白色被毛水貂的1.2515和0.9501倍,但三者间差异不显著(P0.05),相对表达量顺序为:黑色白色灰色。6.基于高通量RNA-seq技术测定了金州黑水貂、银蓝水貂和吉林白水貂皮肤组织转录组,利用qRT-PCR技术对可能影响水貂被毛色素沉积的关键基因进行了mRNA定量表达验证。结果表明,获得毛皮成熟期水貂皮肤转录组原始数据约33.19 G,403,725个Unigene成功注释到7个生物信息学数据库中。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)组合中分别存在12,557、4,334和10,643个差异表达基因,3个组合共有的差异表达基因为1,735个。3个组合中分别存在20、10和17个参与水貂被毛色素沉积的差异表达基因,KITLG、LEF1、DCT、TYRP1、PMEL、TYR、Myo5a、Rab27a和SLC7A11基因可能从黑色素细胞发育、黑素小体成分及其前体、黑素小体转运和真黑色素与褐黑色素合成转变开关这4个生物学过程调控水貂毛色表型。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S865.22
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本文编号：2014686