【作者】 康晓龙；

【导师】 吴常信； 方美英；

【作者基本信息】 中国农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 滩羊是我国优良的地方绵羊品种。主要分布于宁夏中部以及临近甘肃、陕西及内蒙部分区域。滩羊属蒙古羊的一支,具有较强的抗逆性。除产肉性能良好,滩羊主要以生产高品质的滩裘皮而驰名中外。1月龄时期屠宰获得的滩羔皮,被毛洁白亮丽,光泽悦目,毛股纤长呈波形卷曲,花穗美观,轻轻抖动形似波纹涟漪；同时滩裘皮质地轻便柔软,保暖性好,用途较广。随着日龄增加,滩羊的卷曲被毛特征会逐渐减弱甚至消失。基于上述背景,本研究开展以下方面的工作：1)利用RNA-Seq技术分别对1月龄及48月龄时期滩羊皮肤组织进行了转录组测序分析。两个时期皮肤组织共获得了30081个转录本。高表达转录本包括与毛发结构形成相关的TCHH基因以及角蛋白家族成员,KRT25, KRT5, KRT71, KRT14等基因。这些高表达转录本涉及的信号通路参与了毛发的生长发育及结构形成。两个时期的皮肤转录组共获得了37个差异表达基因。差异基因本体注释分析表明部分差异基因在毛发的生长及结构形成中可能具有重要作用。经qPCR验证分析,这些差异基因在两个群体中的表达模式与转录组测序一致,包括SPINK4,KRT1, ESRa, EphA3, ASIP以及GPRl10。同时我们在转录组测序中获得上千个可变剪切事件,信号通路富集结果表明,一些重要的可变剪切事件参与了与毛发生长相关的Notch, VEGF, Wnt, MAPK等通路,而且某些通路只在某一时期特异富集。综合上述因素,我们推测不同时期滩羊皮肤组织转录组测序中的差异基因、可变剪切事件以及其参与的不同信号通路可能在调控滩羊被毛卷曲表型特征方面具有重要作用。这一研究为我们揭示滩羊被毛表型变异及毛发结构特点提供依据,同时该结果也可为研究人类毛发疾病(秃毛症)或毛发结构变异提供了参考信息。2)KRT71是滩羊皮肤组织转录本测序中的高表达基因之一,已报道在其他家养动物卷曲毛发结构形成过程中具有重要作用。基于此,我们研究分析了KRT71基因的结构及调控特征。利用RACE技术成功克隆获得全长为2267bp的绵羊KRT71cDNA序列(登录号为JX975451)。绵羊KRT71基因编码525个氨基酸,具有角蛋白家族特有的模体元件(filement)。无论在核苷酸序列或氨基酸序列与其他物种高度同源,其中与牛同源性最高。表达分析显示,KRT71在包括皮肤在内的不同组织中均表达,但皮肤表达量最高,表明其对动物毛发的生长具有重要作用。不同群体羊皮肤组织KRT71荧光定量PCR结果表明,KRT71在1月龄滩羊皮肤组织表达量高于48月龄的成年羊及1月龄的萨福克羊,但不同群体KRT71序列编码区域无差异,提示KRT71可能在滩羊幼年时期卷曲被毛表型形成过程中具有重要意义。因此,我们对KRT71基因上游2.2kb左右区域的转录调控模式进行了分析。KRT71基因启动子区域的克隆及分析表明转录起始位点前约300bp区域在不同物种间高度保守,含有多个保守转录因子结合位点家族,而经EMSA实验鉴定的CCAAT盒转录因子结合位点可能参与了KRT71基因在不同时期滩羊皮肤组织的差异表达调控过程。上述研究为进一步揭示滩羊卷曲被毛的形成过程具有重要意义,同时对于其他动物毛发结构形态研究也具有参考价值。更多还原

【Abstract】 Tan sheep (Ovis aries), one of the Chinese indigenous breeds, distributes in Ningxia and its adjacent areas. The breed originated from Mongolian sheep amd is hardy and well adapted to a dry, cold and windy environment. It is renowned not only for its excellent meat, but also for the production of high quality pelts. The lamb pelts from Tan sheep are characterized by a natural white color and a lustrous curly fleece. The curly fleece appears when Tan lambs are one month old. After processing, the lamb pelts with curly fleece tend to be thin and light weight, which are well suited for the production of fur coats, carpets, furniture covering, and various forms of handicrafts. However, this unique phenotype disappears gradually with age and the mechanisms behind the phenomenon are still unclearthe. We did experiments below based on the background of Tan lamb curly fleece.1) We identificatified differential expressed genes between adult and lamb Tan sheep by RNA-Seq. In this study, the skin transcriptome data was used for shedding some lights on this issue. In total30,081transcripts including described transcripts and transfrags were identified. The pathway analysis of top100most highly expressed transcripts, which includes TCHH and keratin gene family members, such as KRT25, KRT5, KRT71, KRTI4and others, showed pathways known to be relevant to hair/fleece development and function.37differentially expressed transcripts were detected at two different physiological ages (one-month-old with curly fleece and48-month-old without curly fleece). Subsequent gene ontology analysis showed that a number of genes related to hair/fleece development and showed differential expression between the two physiological stages, the differentially expressed genes (SPINK4, KRTl, ESRa, EphA3, ASIP and GPR110) were confirmed by qPCR analysis. Further pathway analysis of alternative splices tended to be enriched in Notch, VEGF, Wnt, MAPK and some other hair/fleece development related signaling pathways, and some enriched pathways were specifically found in a single physiological stage. We deduced that the differences existed in expressed transcripts, splice isoforms and pathway between two different physiological stages, which might constitute the major reasons for explaining the trait evolvement of curly fleece in Chinese Tan sheep. This study provides some clues for elucidating the molecular mechanism of fleece change with age in Chinese Tan sheep, as well as supplying some potential values for understanding human hair disorder and texture changes.2) The KRT71is one of the highly expressed genes in transcriptome of four samples, which specifically expressed in the IRS (inner root sheath), particular in Henle and Huxley layers of the hair follicle in IRS. Based on clues metioned above and previous data from suppression subtractive hybridization, we cloned and analyzed KRT71gene. KRT71gene has been associated with the curly coat phenotype of animals. The complete cDNA sequence of one ovine gene—KRT71was amplified using rapid amplification of cDNA ends method. The sequence analyses of this novel gene revealed that the sheep KRT71gene encodes a protein of525amino acids which has high homology with the keratin71of other mammal species. Analysis of promoter characters and transcript factors biding site of this gene supply key role for further study. The tissue expression profile analyses indicated that the ovine KRT71gene is generally but differently expressed in the detected tissues including spleen, muscle, skin, stomach, kidney, lung, liver and heart. And the relative expression of KRT71by qRT-PCR was different at Tan sheep with age. Furthermore, the promoter of KRT71gene was conserved at2.2kb before transcription start site among species, and had some conserved transcription factor binding site families. And the CCAAT box might have some important function in regulating the differential expression of KRT71in Tan sheep skin tissues at different stages. The cloning and character analysis of the sheep KRT71gene will probably provide molecular basis for associating KRT71gene with curly fleece phenotype of Tan sheep, as well as other animals with curly coat.更多还原