绵羊WNT5A基因克隆及其组织表达分析

Analysis of WNT5A Gene Cloning and Tissue Expression in Sheep

WNT5A(Wnt family member 5A)参与了多种细胞的增殖、凋亡和分化等生物学过程,在乳腺形态发生、毛囊发育等方面发挥了重要作用。该试验利用RT-PCR获得绵羊WNT5A基因的CDS区,分析WNT5A蛋白的结构特征并利用RT-qPCR检测WNT5A基因在9个组织中的表达情况。绵羊WNT5A基因的CDS区全长1062 bp,编码353个氨基酸。该蛋白的二级结构主要以α-螺旋和无规则卷曲为主,β转角、延伸链贯穿于整个蛋白质结构中。String分析结果表明,WNT5A与卷曲蛋白受体、低密度脂蛋白相关蛋白和受体酪氨酸激酶有相互作用。RT-qPCR研究结果表明,WNT5A基因表达具有组织特异性及品种特异性。WNT5A基因在绵羊心脏、肝脏、肺脏、脾脏、肾脏、卵巢、背最长肌、乳腺和皮肤9个组织中均广泛表达,其中在皮肤中的表达量高于其他组织(P<0.05),而在脾脏中表达量较低。它在甘肃高山细毛羊皮肤、乳腺、背最长肌、肾脏、肺脏、脾脏和肝脏中的表达量高于小尾寒羊(P<0.05),而在甘肃高山细毛羊卵巢中的表达量低于小尾寒羊(P<0.05),在心脏中该基因的表达量无差异(P>0.05)。该结果为深入研究绵羊WNT5A基因的功能提供了重要依据,同时也丰富了绵羊基因组内容。

Wnt family member 5A is involved in the biological processes of proliferation,apoptosis and differentiation of various cells,and plays an important role in mammary morphogenesis and hair follicle development.The Coding sequence region of sheep WNT5A gene was obtained by reverse transcription-polymerase chain reaction and the structural characteristics of WNT5A protein were analyzed using bioinformatics.The expression of WNT5A gene in nine tissues was detected by real time-quantitative polymerase chain reaction.The coding sequence region of sheep WNT5A gene was 1062 bp in length which is encoding 353 amino acids.The secondary structure of the protein is mainly composed of α-helix and random coil,and the β-turn and extension chain penetrate through the entire protein structure.String analysis revealed that WNT5A interacts with frizzled receptor,low density lipoprotein related protein,and receptor tyrosine kinase.The results of real time-quantitative polymerase chain reaction showed that WNT5A gene expression was tissue specific and variety specific.In the study,WNT5A gene was widely expressed in nine tissues,and its expression in skin was higher than other tissues(P<0.05),the expression level was lower in the spleen;the expression level in the skin,mammary gland,longissimus dorsi,kidney,lung and liver of Gansu alpine merino sheep was higher than that of small-tail Han sheep(P<0.05).The expression level of ovary in Gansu alpine merino sheep was lower than that in small-tail Han sheep(P<0.05),and there was no difference in the expression of this gene in the heart.This result provides an important basis for further study of the function of sheep WNT5A gene,and also enriches the content of sheep genome.