中国美利奴超细型与哈萨克羊毛囊兴盛期皮肤组织消减cDNA文库的构建

Construction of Subtractive cDNA Library of Skin Tissue in Follicle Anagen Between Chinese Merino and Kazakh Sheep

【目的】筛选影响绵羊羊毛性状的重要候选基因,为研究羊毛性状形成的分子机制奠定基础。【方法】利用抑制性消减杂交技术构建中国美利奴超细型和哈萨克羊兴盛期皮肤组织间正、反向消减cDNA文库,并对差异基因进行测序和生物信息学分析;应用半定量RT-PCR和实时荧光定量RT-PCR对部分差异基因进行鉴定。【结果】①从所构建绵羊皮肤组织的正、反向消减cDNA文库中分别获得153和143个ESTs,其中分别包括5和4个未知功能的ESTs,推测可能是新基因;对部分已知功能ESTs进行了GO聚类分析、通路分析和蛋白互作分析,结果显示粗、细毛羊之间存在一定差异;②文库中的3个差异基因KRTAP8-2、Trichohyalin和KRTAP3-2均能在皮肤中特异性地高表达,其中KRTAP8-2和Trichohyalin基因在中国美利奴超细型羊皮肤组织中的表达丰度分别是哈萨克羊的3.45和2.07倍,而KRTAP3-2基因在哈萨克羊皮肤组织表达丰度是中国美利奴超细型羊的1.87倍。【结论】成功构建了中国美利奴超细型和哈萨克羊皮肤组织间抑制性消减cDNA文库,初步筛选出一批可能影响羊毛性状的差异表达ESTs。

[ Objective ]The aim of the study is to screen important candidate genes influencing wool traits in sheep,and to lay a theoretical foundation for exploring the molecular mechanism of wool traits formation.【Method】Using suppression subtractive hybridzation（SSH） technique,positive and reverse subtracted cDNA library between skin tissue in follicle anagen of Chinese Merino（superfine type） and Kazakh sheep was constructed,respectively.Genes of differential expression profiles were sequenced for bioinformatics analysis.Semi-quantitative RT-PCR and real-time fluorescent quantitative RT-PCR methods were used to assess partial genes of differential expression profiles.【Result】 A total of 153 and 143 ESTs were obtained from the forward and reverse subtracted cDNA library,respectively.Among these ESTs,5 from positive library and 4 from reverse library of unknown function were identified and speculated as novel genes.The GO cluster,pathway and protein-protein interaction analysis of the partial ESTs of known function showed that there were certain differences between coarse wool and fine wool sheep.The three differential genes from the constrcted cDNA libraries,KRTAP8-2,Trichohyalin and KRTAP3-2 genes were all specifically expressed at high level in the skin.The abundance of KRTAP8-2 and Trichohyalin genes mRNA in skin of Chinese Merino was 3.45 and 2.07 times than that of Kazakh sheep,respectively,while the abundance of KRTAP3-2 gene mRNA in skin of Kazakh sheep was 1.87 times than that of Chinese Merino.【Conclusion】The subtracted cDNA library of skin tissue between Chinese Merino and Kazakh sheep was successfully constructed,a batch of ESTs with differential expression profiles which possibly influence wool traits were initially screened.