绵羊角细胞关联蛋白2基因克隆与表达分析

The Cloning and Expression Analysis of Keratinocyte-associated Protein 2 Gene from Sheep

为克隆绵羊角细胞关联蛋白2(Keratinocyte-associated protein 2,KRTCAP2)基因mRNA并分析该基因表达分布规律,本研究首先提取小尾寒羊与新吉细毛羊皮肤及不同组织总RNA,RT-PCR方法克隆KCTCAP2基因并进行两个品种间的比较分析,随后利用HRM方法对目的SNP位点进行基因多态性分析,最后用q RT-PCR方法分析两个品种不同组织间KRTCAP2基因的时空表达分布。RT-PCR扩增显示绵羊皮肤组织出现略小于500 bp的特异性条带,测序证实该片段为KRTCAP2基因,长度为466 bp,编码149个氨基酸。所克隆的4条片段中存在4个SNP位点,其中2个SNP位点引起氨基酸位点突变。针对c.194位A>G突变位点设计的HRM检测发现小尾寒羊、新吉细毛羊等群体中并不存在该多态位点。小尾寒羊与新吉细毛羊不同组织间的表达分布显示,KRTCAP2基因存在多组织表达特性,在两品种羊组织表达模式既存在一致性也存在差异性。一致性主要表现为小尾寒羊与新吉细毛羊中肝脏、脾脏、肺脏、肾脏、肠道与皮肤组织中KRTCP2表达量较心脏组织呈现上调表达的趋势。差异性表现为新吉细毛羊在肝脏、脾脏、肺脏、肠道、脑和皮肤的组织表达量均高于小尾寒羊,在肌肉和卵巢组织中的表达量低于小尾寒羊,在肾脏组织中的表达量基本持平。本研究成功克隆出绵羊KRTCAP2基因并进行了系统分析,为探讨绵羊角细胞功能奠定了基础。

In order to clone the Keratinocyte-associated protein 2(KRTCAP2)gene and identify the expression pat-tern in different organs of sheep,in this study,the total RNA was extracted from different organs coming from Smalltail Han sheep(STH sheep)and Xinji Fine wool sheep(XJ sheep),and KRTCAP2 was cloned by RT-PCR,the poly-morphisms in different sheep populations were tested by HRM.In the end,the tissue expression pattern was mea-sured by q RT-PCR.The RT-PCR amplification showed there was a specific band which was slightly smaller than500 bp,and the sequencing data approved the amplicons were KRTCAP2 gene with 466 bp in length encoding 149 amino acids peptides.In the same time,four SNP mutants were identified,and two of them caused mutation in pro-tein.The polymorphism of A>G mutant in c.194 by HRM analysis showed there were no polymorphism in all popula-tions including STH sheep and XJ sheep.The tissues expression distribution analysis by q RT-PCR approved thatKRTCAP2 gene was constructive expressed in all organs in sheep.However,the expression pattern has both similarand distinct point in organs from STH sheep and XJ sheep.The similar point is KRTCAP2 has higher expression inliver,spleen,lung,kidney,gut and skin than heart in both STH sheep and XJ sheep,The expression levels in liver,spleen,lung,intestine,brain and skin were higher in XJ sheep than in STH sheep.The expression in muscle and ova-ry tissue of XJ sheep was lower than that of STH sheep,and the expression in kidney tissue was lower.In conclusion,the sheep KRTCAP2 gene was cloned in this study which provides an opportunity to explore the gene functionin further.