绵羊角蛋白关联蛋白8.2基因克隆与表达分析

Cloning and Expression Pattern Analysis of KAP8.2 Gene in Sheep

本研究旨在克隆绵羊角蛋白关联蛋白8.2(keratin-associated protein,KAP8.2)基因mRNA并分析该基因在不同组织器官的表达分布。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KAP8.2基因并进行两个品种间的比较分析,实时荧光定量PCR方法分析两个品种间KAP8.2基因的表达谱差异。结果表明已成功克隆出绵羊KAP8.2基因mRNA,该片段长574bp,其中ORF区192bp,编码63个氨基酸,甘氨酸(Gly)和酪氨酸(Tyr)含量依次为23.8%和20.6%,属于典型的HGT-KAP家族。多态性分析显示新吉细毛羊与小尾寒羊KAP8.2基因间存在丰富的多态性,多态位点多位于CDS区两侧,其中小尾寒羊KAP8.2基因3′UTR区c192+19-39出现一个疑似fru-let-7j的作用靶位。不同组织表达谱分析显示该基因为多组织表达基因,但两个品种羊不同组织表达谱存在较大差异,表现为小尾寒羊在脾脏、肝脏和皮肤中高表达,而新吉细毛羊则在皮肤和心脏中高表达。本研究提示小尾寒羊与新吉细毛羊KAP8.2基因在基因多态性还是组织表达分布上存在显著差异,提示该基因与毛表型性状相关。

The object of this study was to clone the Keratin-associated protein 8.2（KAP8.2）gene from Small-tail Han sheep and Xinji Fine-wool sheep and test the tissues expression pattern.In this study,the total RNA were extracted from different tissues firstly,and KAP8.2gene was cloned by RT-PCR.In the end,the different expression patterns of KAP8.2gene in Small-tail Han sheep and Xinji Fine-wool sheep was detected by QRT-PCR.The cloning results showed that the KAP8.2gene were cloned,and the length of KAP8.2mRNA was 574 bp,containing a 192 bp ORF which encoded a protein of 63 amino acids.These KAP8.2gene belonged the typical HGTKAP family because the rate of glycine and tyrosine were 23.8% and 20.6%,respectively.The polymorphism analysis showed that there were some polymorphisms in KAP8.2gene and almost SNPs located on both sides of CDS region.The region which located on the 3′UTR c192＋19-39 of KAP8.2from Small-tail Han sheep might be the target of fru-let-7j.The expression pattern analysis showed that KAP8.2gene was a multiple organs expression gene,and there were signifi-cant differences in expression patterns of KAP8.2gene in Small-tail Han and Xinji Fine-wool sheeps.In Small-tail Han sheep,KAP8.2gene was over-expressed in spleen,liver and skin,but in Xinji Fine-wool sheep,KAP8.2gene was over-expressed in skin and heart.In conclusion,there were significant differences in gene polymorphisms and expression pattern between Small-tail Han sheep and Xinji Fine-wool sheep,and the result suggested the KAP8.2gene associated with some phenotypes of wool.