中国美利奴羊皮肤POU2F3基因及其剪接体的克隆与表达分析

Cloning and Expression Characterization of Sheep POU2F3 Gene and Its Isoforms in Chinese Merino Skin

旨在研究绵羊POU2F3(POU class 2homeobox 3)基因的组织表达规律,并克隆中国美利奴羊(新疆军垦型)POU2F3基因多种剪接体的全长编码区(Coding sequence,CDS区)。采用半定量RT-PCR方法分析绵羊POU2F3基因的组织表达特性;采用RT-PCR扩增绵羊POU2F3基因的全长CDS区,克隆后进行测序分析。组织表达谱分析表明,POU2F3基因仅在绵羊部分内脏器官和组织中表达,其中在皮肤组织中的表达量较高;POU2F3基因在超细毛品系羊体表不同部位皮肤组织中的表达水平差异不显著(P>0.05),在细毛羊和粗毛羊体侧皮肤中的表达量也不存在显著性差异(P>0.05);序列分析结果表明,POU2F3基因在绵羊皮肤组织中至少存在4种mR-NA剪接形式,相应的开放阅读框(Open reading frame,ORF)长度依次为1 290、1 191、666和666bp,分别编码429、396、221和221个氨基酸。中国美利奴羊(新疆军垦型)POU2F3基因在皮肤组织中具有较高表达水平;绵羊POU2F3基因在皮肤组织中除了全长的POU2F3蛋白外,至少还有2种不同的蛋白剪接体。本研究为阐明绵羊POU2F3基因的功能以及开展优质细毛羊的培育工作奠定了基础。

The objective of this study was to explore the expression pattern of POU2F3 gene in sheep,and to clone the full-length CDS of POU2F3 and its alternatively spliced isoforms in Chinese Merino skin（Xinjiang Junken Type）.The expression of POU2F3 gene was detected by Semi-quantitative reverse transcription PCR（SqRT-PCR） in tissues.The full-length CDS regions of POU2F3 gene and its isoforms were amplified by RT-PCR,cloned into T vector,and sequenced.The sequence was analyzed using online bioinformatics softwares.SqRT-PCR analysis showed that the POU2F3 gene was only expressed in some sheep internal organs and tissues.Comparatively,it was highly expressed in body side skin.No significant differences at the expression levels of POU2F3 gene between the skins from various anatomic locations of super fine wool strain were observed（P0.05）.Similarly,no significant differences at the expression levels of POU2F3 gene in body side skin between wool and hair sheep were observed（P0.05）.Sequence analysis indicated that sheep POU2F3 gene in skin generated at least four spliced mRNA isoforms encoding four POU2F3 protein isoforms of 429,396,221 and 221aa,respectively.POU2F3 gene is highly expressed in the skin of Chinese Merino（Xinjiang Junken Type）.Besides the full-length POU2F3,sheep POU2F3 has at least two protein isoforms in skin.This study not only lays the foundation for clarifying the function of sheep POU2F3 gene,but also provides theoretical basis for breeding high quality Merino sheep.