【作者】 杨晓光；

【导师】 郑冬；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2015， 博士

【摘要】 鹿茸角是唯一每年周期性表形态再生的哺乳动物附件器官,即远端附件去除后还会完整地再生出来。因此鹿茸为研究再生的分子机理提供了一个独有的动物模型。鹿茸角一个再生周期包括四个阶段：快速生长期,钙化期,茸皮(也称为鹿茸)脱落期,以及硬角脱落期。在快速生长期,鹿角由软骨和骨组成,渗透以血管和神经网络,外覆茸皮。快速纵向生长始于每个鹿茸角顶端增生区。在此增生区内,在鹿茸软骨膜(AP)内的祖细胞进行增殖和分化,向外形成包于外部的茸皮,向内形成排列于垂直小梁上的软骨前体细胞和软骨细胞。鹿茸角具独有的茸性上皮,上分布有稀疏的绒毛,被称为鹿茸(茸皮)。研究表明,茸皮并不是包裹于角柄外典型头皮在鹿茸角部分上简单的延伸。茸皮再生始于包裹角柄创面皮肤的伤口愈合过程。但是其特殊性在于,当创伤愈合上皮迁移穿过角柄骨膜(PP)远端点后,皮肤从头皮类型开始转换成茸皮类型。相较于角柄皮肤(典型的头皮),茸皮缺乏皮下疏松结缔组织层,但有更厚的表皮层,并能形成新的毛囊。这些新形成的毛囊缺乏立毛肌和汗腺,但具有较大皮脂腺。在增生区向内侧,虽然鹿茸软骨基质生物化学性质上类似于其他透明软骨,其独特之处在于具有血管网络。上述组织学特性使茸皮和软骨组织与其他组织显著不同,其中通过一系列的增殖、成型、分化事件,基因表达程序发生大规模的变化来形成新的结构。然而,我们对其转录组信息知之甚少。本论文对快速生长期马鹿(Cervus elaphus)茸皮和软骨进行了研究,通过自体转录组组装,并对构成55,095,730条干净短reads(测序序列)的总计长度为4,958,615,700核苷酸的序列进行了RNA-Seq分析。自体组装序列利用BLASTX与NCBI nr、GO、 COG、KEGG数据库进行比对和标注。结果表明：1. 利用Trinity软件自体组装共获得63,550条All-Unigenes。其中,两种组织共有的All-unigenes为33,295条,茸皮组织特有的All-unigenes为20,139条,软骨组织特有的All-unigenes为10,116条。2.通过nr注释,有33,471条All-Unigenes可以直接比对到Nr数据库,占All-unigenes总数比例为52.67%；32,762条All-Unigenes比对到Swissport数据库(51.55%)；25,577条All-Unigenes比对到KEGG数据库；11,428条All-Unigenes比对到COG数据库(17.98%)；此外,还有9,252条All-Unigenes比对到GO数据库(14.56%)。其余序列用ESTscan软件进行编码区预测,有691条可能为新的蛋白编码序列。3.功能分类及代谢通路分析结果表明,在茸皮和软骨的生长发育过程中,参与细胞、细胞骨架、核糖体结构、细胞外基质等构成、核酸与蛋白质生物合成转运、翻译、催化活性和代谢过程、细胞增殖调控、抗细胞凋亡等相关基因和通路起到了重要调控作用。4.从茸皮转录组数据中共挖掘得到44种生长因子及26种生长因子受体。其中IGF2表达量最高。利用qPCR法对8种随机选取的生长因子或受体进行基因表达水平的验证。结果表明qPCR法与转录组分析结果基本一致。5.茸皮和软骨各自特异性表达6961和2776条基因。在两种组织共同表达基因中,有7,966条基因表达差异显著(|log2Ratio|≥1且FDR≤0.001)。其中,茸皮较软骨有5,779条All-unigenes表达显著上调,而软骨较茸皮有2,187条基因表达显著上调。通过GO功能富集分析及KEGG通路富集分析,这些差异表达基因主要参与细胞结构、细胞代谢、蛋白质相互作用、催化活性等生物学进程。其中涉及注释了5,328个基因的236条通路。上述数据结果是目前关于马鹿鹿茸角顶端茸皮和软骨再生最全面的基因序列。将为进一步研究鹿茸分子遗传学和功能基因组学提供依据。更多还原

【Abstract】 Deer antlers are the only mammalian appendages that are subject to an annual cycle of full renewal. It provides a unique model to explore the mechanisms underlying epimorphic regeneration, a phenomenon of de novo development of appendages distal to the level of amputation in mammals. The yearly renewal cycle of antlers briefly consists of four stages: rapid growth, calcification, antler skin (also called velvet) shedding and antler casting. During the rapid growth stage, antlers are composed of cartilage and bone, infiltrated with blood vessels and nerves networks and covered by a velvet skin. Rapid longitudinal elongation occurs at the growth zone located in the distal tip of each antler branch. Within this growth zone, progenitor cells in the antler perichondrium (AP) proliferate and differentiate externally into vevlet skin, and internally into chondroprogenitors and chondrocytes arranged in vertical trabeculae.Antlers have a unique velvet-like skin that is sparsely populated with hair and is known as velvet, However, studies have shown that antler external velvet components are not simple extensions of typical scalp skin enveloping pedicles. Regeneration of velvet skin commences with the wound healing over a pedicle stump by distal pedicle skin. The uniqueness in this case is, however, that when the centripetally migrating healing skin passes the point of distal pedicle periosteum (PP), the skin begins to change in nature from scalp type to velvet type. In contrast to pedicle skin (a typical scalp skin), antler velvet lacks a subcutaneous loose connective tissue layer, but has a much thicker epidermis and the ability to form new hair follicles. These newly formed follicles lack associated arrector pili muscles and sweat glands but possess large multilobed sebaceous glands. On the internal side, although the matrix of antler cartilage is biochemically similar to that of other hyaline cartilages, its unique feature is an extensive vascular network. These histological features make velvet and cartilage unique tissues types, underlying which large-scale changes in gene expression programs occur to create the new structures, through a series of proliferation, patterning, and differentiation events. However, little transcriptomic information is available for them.In this study, for velvet skin and cartilage from the antler of Red deer (Cervus elaphus), we performed de novo transcriptome assembly and analysis of total 4,958,615,700 nucleotides composed 55,095,730 clean short reads using RNA-Seq technology. After de novo assembly, with BLASTX alignment, assembled sequences were then annotated to NCBI nr, Gene ontology terms, Clusters of Orthologous Groups classifications, and Kyoto Encyclopedia of Genes and Genomes pathways databases. The results showed:1. De novo assembly of the clean reads by Trinity resulted in 63,550 unique all-unigenes. Velvet skin and catilage shared 33,295 all-unigenes,20,139 all-unigenes were vevlet-specific, and 10,116 were specially from cartilage.2. After blasted against the NCBI nr database.33,471 genes (52.67% of all unigenes) returned a BLAST result above the cut-off value (E-value 10-5). Similarly, up to 32,762 all-unigenes (51.55% of all unigenes) had Swissprot annotation, and 11,428 all-unigenes (17.98% of all unigenes) had COG annotation. Based on GO classifications,9,252 sequences (14.56%) were categorized into 52 functional groups. Meanwhile,25,577 all-unigenes (40.25%) were blasted into 241 KEGG pathways. All-unigenes have no hit in blast were predicted by ESTScan,691 putative novel genes were found and translated into peptide sequencs.3. Functional annotation and metabolic pathways analysis disclosed, during velvet and cartilage growth and development at the rapid growth stage, genes and pathways, involving cell, cellular skeleton, ribosomal structures, extracellular matrix, nucleitides and protein biosynthesis and transfer, translation, catelytic activity. And metabolic process, cell proliferation regulation, anti-apoptosis, play critical regulation functions.4. Through data mining,44 kinds of growth factors and their 22 kinds of receptors were discovered in antler velvet skin. Among them, insulin-like growth factor 2 had the highest expression level. Six growth factors and two kinds of recptors were selected and validated by real-time qPCR method.5. Anlter velvet skin and cartilage owned 6,961 and 2,776 tissue-specific genes, respectively. Within the shared genes,7,966 showed differentitial expression (log2Ratio≥1 and FDR≤0.001). Among them,5779 genes were up-regulated in velvet skin, and 2187 genes were up-regulated in cartilage. Through GO and KEGG enrichment analysis, these differential genes focused on cell components, cellular metabolics, proteins interaction, catalytic activity. 5328 genes involved were blasted against 236 pathways.These data represents the most comprehensive sequence resource available for the regenerating antler velvet skin and cartilage at the Red deer antler growth tip. and provide a basis for further research on deer antler molecular genetics and functional genomics.更多还原