山羊Eda基因的克隆、序列分析及表达

【目的】克隆山羊Eda基因CDS区全序列，并对其进行序列分析，研究Eda基因在毛囊生长周期不同阶段皮肤组织中的表达规律。【方法】以太行黑山羊为研究对象，采集其毛囊生长休止期、生长期和退行期背部皮肤组织，采用RT-PCR技术克隆山羊Eda基因，通过在线软件Blastn进行基因cDNA序列分析，用SMART进行氨基酸序列分析，利用SWISS-MODEL软件进行蛋白质结构分析；以β-actin为看家基因，采用实时荧光定量PCR技术对 mRNA在毛囊生长不同时期皮肤组织中的表达规律进行分析。【结果】太行黑山羊Eda-A1基因CDS区全长1 176 bp，编码391个氨基酸；Eda-A2比Eda-A1少6个碱基（nt1161-1166），编码389个氨基酸；山羊Eda蛋白氨基酸序列相似性在不同物种间均大于90%。SMART分析表明，CDS编码的蛋白质包含了TNF、跨膜区以及胶原等结构域。SWISS-MODEL软件预测结果表明，Eda-A2与Eda-A1在蛋白质表面结构上存在明显差异。毛囊退行期的皮肤组织中，Eda mRNA表达量最高，且极显著高于毛囊休止期和毛囊生长期（P＜0.01），毛囊生长期的表达量中等且显著高于休止期（P＜0.05），毛囊休止期的表达量较低。【结论】Eda基因CDS区在物种间保守性较强，Eda mRNA在毛囊生长周期不同阶段表达量有差异，推测Eda基因对毛囊生长周期的调控有一定影响。

Molecular cloning,sequence analysis and expression of goat Eda gene

【Objective】 The objective of the present study was to obtain and analyze CDS sequence of goat Ectodysplasin-A gene(Eda),and to investigate the expression patterns of Eda in key stages of hair follicle cycles.【Method】 The dorsal skin biopsys were surgically collected from three adult male Taihang black goats at telogen,anagen and catagen.The CDS of Eda gene were obtained by RT-PCR.The expression of Eda mRNA in different stages of hair follicles cycle was analyzed by real-time quantitative PCR.The β-actin was used as housekeeping gene.【Result】 The size of Eda-A1 CDS was 1 176 bp encoding 391 amino acid residues.The Eda-A2 was 6 bases(nt1161-1166) shorter than Eda-A1 encoding 389 amino acid residues.The amino acid sequences shared more than 90% identity with other Eda species.Analysis by SMART suggested that the encoded protein contained TNF motif,collagen and transmembrane segment.Analysis with SWISS-MODEL suggested that the surface shape of the Eda-A2 protein showed distinct difference compared to Eda-A1.The Eda mRNA levels in skin were most abundant at categen,and were significantly higher than that at anagen and telogen(P<0.01).The expression levels were medium at anagen,and were significantly higher than that at telogen(P<0.05).The expression levels were lowest at telogen.【Conclusion】 The result indicates that Eda gene is highly conservative among species,and expression of Eda mRNA fluctuates in a hair cycle-dependent manner.It is inferred that Eda gene may be involved in molecular regulation of hair cycle.