【作者】 姜维；

【导师】 陈玉林；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2012， 博士

【摘要】 山羊绒是优良的纺织原料，在国际上享有“软黄金”的盛誉。山羊绒是皮肤组织的衍生物，发生发育并分化于皮肤的次级毛囊中。研究表明，山羊的次级毛囊每年有生长期、退行期、休止期的周期性变化。毛囊生长周期的变化是一个复杂的生理过程,受一系列信号分子调控。因此，寻找与发现次级毛囊生长期-退行期转化过程的调节分子是研究山羊绒品质改良的重要基础。在毛囊生长发育过程中，毛囊生长期的持续时间直接决定了毛干的长度，寻找毛囊退行期诱导的调控分子，分析毛囊生长期-退行期转化过程中涉及的调控因子，对于研究山羊绒长度性状相关的功能基因具有特殊的意义。在过去十年里，研究者以老鼠作为实验动物，通过研究毛囊生长周期缺陷小鼠，及生长周期不同阶段基因的表达模式，已经鉴定出了多个调控分子。然而，山羊次级毛囊生长期向退行期转化过程中调控因子的表达及特点，相关的研究很少。本研究利用抑制消减杂交技术，构建了太行黑山羊毛囊生长期与退行期早期皮肤组织正反向cDNA消减文库，并对正向文库中的皮肤组织差异表达基因进行了筛选、鉴定和分析，取得如下主要结果：1.利用SSH技术构建了太行黑山羊毛囊生长期与退行期早期皮肤组织正向（以退行期早期皮肤组织为TestercDNA；以生长期皮肤组织为drivercDNA）和反向cDNA消减文库（以生长期皮肤组织为TestercDNA；以退行期早期皮肤组织为drivercDNA），用看家基因GAPDH（甘油三磷酸脱氢酶）检测出两个cDNA文库的消减效率分别为211和28，表明文库中非差异表达基因得到了有效的消减。2.利用巢式PCR扩增及斑点杂交技术对已建立的正向文库进行了筛选。基于3000个克隆子，经过两轮严格筛选，获得了72个阳性克隆。经测序分析获得了45个非冗余ESTs。其中，42个ESTs是山羊的未知序列，经分析其与绵羊、牛等其它家畜的相似性≧84%；2个ESTs是山羊的已知序列；另外1个EST（TA2212）在NCBI数据库中尚未找到其相似性序列，属首次发现的基因。3.利用实时荧光定量PCR技术对消减文库中获得的重复次数较多的6个与毛囊发育相关的基因及首次发现的未知序列TA2212进行了验证。结果表明这7个基因在太行黑山羊退行期早期皮肤组织中的表达量较生长期皮肤组织中的分别提高：2.79（TA2212）、2.53（Eda）、1.64（BMPR1A）、1.59（MSTN）、1.56（Catenin）、1.42（TP53INP1）和1.41（IGFBP3）倍，说明这些基因在毛囊生长期向退行期过渡时发挥着上调作用。4．对毛囊生长期-退行期过渡时期mRNA表达量增幅较大的未知序列TA2212进行RT-PCR验证，并对该序列特征进行了分析，同时，采用实时荧光定量PCR技术，检测了毛囊生长周期中不同发育时期皮肤组织中TA2212的mRNA表达量。结果表明：TA2212EST长1068bp，第16-171号核苷酸为一完整的ORF，SMART分析发现该序列编码一个包含26个氨基酸残基的末端信号肽序列；实时荧光定量分析显示：毛囊休止期时，TA2212的mRNA表达量较低（0.0933±0.0189）；毛囊生长期时，其mRNA表达量下降了约12%（0.0827±0.0078）且与休止期差异不显著（P>0.05）；毛囊退行期时，其mRNA表达量提高了约3.3倍（0.3107±0.0216）且极显著高于毛囊休止期和毛囊生长期（P<0.01）。5.对毛囊生长期-退行期过渡时期mRNA表达量增幅较大的BMPR1A基因进行了克隆，并对该基因CDS区核苷酸序列及其编码的蛋白质序列进行序列特征分析，同时，采用实时荧光定量PCR技术，检测了毛囊生长周期中不同发育时期皮肤组织中BMPR1A基因mRNA的表达量。结果表明：该基因的CDS序列长1599bp，编码532个氨基酸；其核苷酸序列及其预测的氨基酸序列与其他物种相比，序列相似性在92%以上；其ORF编码的蛋白质经SMART程序分析具有信号肽、Activin_recp、跨膜结构域、GS、以及S_TKc结构域等五个保守功能结构域位点；实时荧光定量分析显示：毛囊休止期时，BMPR1A基因mRNA的表达量较高（0.1121±0.0194）；毛囊生长期时，其mRNA表达量下降了约41%（0.0663±0.0023）且与休止期差异不显著（P<0.05）；毛囊退行期时，其mRNA表达量上升了14%（0.0756±0.0113）且与毛囊休止期和毛囊生长期时差异不显著（P<0.05）。6.对毛囊生长期-退行期过渡时期mRNA表达量增幅较大的Eda基因进行了克隆，并对该基因CDS区核苷酸序列及其编码的蛋白质序列进行序列特征分析，同时，采用实时荧光定量PCR技术，检测了毛囊生长周期中不同发育时期皮肤组织中Eda基因mRNA的表达量。结果得到太行黑山羊Eda-A1和Eda-A2基因，其中Eda-A1基因编码区序列全长1176bp，编码391个氨基酸，Eda-A2基因编码区序列全长1170bp，编码389个氨基酸，Eda-A2比Eda-A1仅缺少6bp（ntll61-1166）；太行黑山羊Eda-A1及Eda-A2基因核苷酸序列及其预测的氨基酸序列与其他物种相比，序列相似性在90%以上；其ORF编码的蛋白质经SMART程序分析具有TNF、跨膜区以及由19个G-X-Y重复形成的胶原等保守功能结构域位点；其编码的蛋白质经SWISS-MODEL程序分析发现Eda-A2和Eda-A1在蛋白质表面结构上存在明显差异；实时荧光定量分析显示：毛囊休止期时，Eda基因的mRNA表达量较低（0.0158±0.0014）；毛囊生长期，其mRNA表达量提高了约1.9倍（0.0302±0.0021）且显著高于休止期（P<0.05）；毛囊退行期时，其mRNA表达量上升了约7倍（0.2066±0.0266）且极显著高于毛囊休止期和毛囊生长期（P<0.01）。更多还原

【Abstract】 Cashmere is an excellent textile material, a derivative of skin tissue, and enjoys a “softgold” reputation in the international community. Its development and differentiation occurs inthe secondary follicles in goat skin. Studies have shown that goat secondary follicle presentscyclical changes with seasons, turns into the anagen, catagen and telogen. Recent researchesfind that these properties have associated with a unique pattern of genes expression, involvingcomplex regulatory mechanisms. Particular attention has been paid to anagen-catagentransition regulatory molecules. Because the lengths of cashmere were determined by theduration of hair follicle anagen, it is very meaningfull to find the regulators involving insecondary hair follicle anagen-catagen transition. In mammals, the mouse has been used asthe model for the study of these regulatory genes. Over the past decade, many of theanagen-catagen transition regulatory molecules were identified. However, little is knownregarding the characteristics of these genes and their expression pattern in goat. In this study,we generated a library enriched in up-regulated ESTs at anagen-catagen transition of Taihangblack goat secondary hair follicle using suppressive and subtractive hybridization (SSH), anddifferentially expressed ESTs from forward SSH library was confirmed and analyzed. Themain results were as follows:1. Both the forward (tester: the cDNA from skin during early catagen, driver: thecDNA from skin during late anagen) and reverse (tester: the cDNA from skin during lateanagen, driver: the cDNA from skin during early catagen) SSH analyses were conducted.The subtraction efficiency was estimated by a housekeeping gene GAPDH, and the resultshowed that GAPDH was subtracted efficiently by211and28folds for forward and reversesubtracted cDNA library respectively, which indicated that the reduction of GAPDH cDNAwas observed in the subtracted library.2. The products from forward SHH were cloned. The clones were were selected forfurther PCR amplifications with the nest primers and screened using dot blotting assay. Ourstringent judgment led to the selection of72positive clones from3000clones. Aftersequencing and analysis, forty-five high-quality sequences were aligned to the GenBankdatabase. forty-two clones were unknown in goats but have high homology with Ovis aries, Bos taurus or other species with identity over84%, Two clones were known in goats, andone clone was found without any significant matches with the GenBank database..3. Six genes that occured more frequently in library and the potential novel gene wereselected for quantitative real-time PCR validation assay to confirm the expression profilesgenerated by SSH. The result showed that their expression quantity varied from one to threefolds (2.79(TA2212),2.53(EDA),1.64(BMPR1A),1.59(MSTN),1.56(Catenin),1.42(TP53INP1) and1.41(IGFBP3)), indicating that those genes increasingly expressed in theanagen-catagen transition.4. The potential novel gene, TA2212, was successfully amplified by RT-PCR and thesequences were analyzed. The expression of TA2212in different stages of hair folliclescycle was analyzed by real-time quantitative PCR. An open reading frame of171bp wasfound at16-171nucleotides. Analysis by SMART suggested that a26amino acid residue ofterminal signal peptide was found. The real-time quantitative expression analyses showedthat the TA2212was expressed at a lower level during telogen (0.093296±0.018871) and itsexpression decreased by12%during anagen and it had no significant difference (P>0.05)compared to telogen (0.082684±0.007827). Its expression increased by3.3folds duringearly catagen (0.310684±0.021578) and it was significantly higher than the anagen andtelogen (P <0.01).5. The BMPR1A gene was cloned and the sequences were analyzed. The expression ofBMPR1A mRNA in different stages of hair follicles cycle was analyzed by real-timequantitative PCR. We found the size of BMPR1A CDS was1599bp encoding532aminoacid residues. The amino acid sequences shares more than92%identity with other speciesBMPR1A. Analysis by SMART suggested that the encoded protein contained signal peptide,Activin_recp motif, GS, transmembrane segment and S_TKc domain. The BMPR1A mRNAlevels in skin were abundant at telogen (0.112077±0.01942). The expression levelsdecreased by41%at anagen (0.066293±0.002288) and it had no siginificant difference(P>0.05) compared to the telogen. The expression levels increased by14%at categen and ithad no siginificant difference (P>0.05) compared to the telogen and the anagen.6. The Eda gene was cloned and the sequences were analyzed. The expression of EdamRNA in different stages of hair follicles cycle was analyzed by real-time quantitative PCR.We found the size of Eda-A1CDS was1176bp encoding391amino acid residues; TheEda-A2was only6bases (nt1161-1166) shorter than Eda-A1encoding389amino acidresidues. The amino acid sequences shares more than90%identity with other species Eda.Analysis by SMART suggested that the encoded protein contained TNF motif, collagen andtransmembrane segment. Analysis with SWISS-MODEL suggested that the surface shape of the Eda-A2protein showed distinct difference compared to Eda-A1. The expressionlevels were lowest at telogen (0.015793±0.001407). The expression levels increased by1.9folds at anagen (0.030191±0.002114) and were significantly higher than that at telogen(P<0.05). The Eda mRNA levels in skin increased by7folds at categen(0.206635±0.026615) and were significantly higher than that at anagen and telogen更多还原