梅花鹿天然Toll样受体9基因克隆与序列分析

Trizol法提取梅花鹿肝脏总RNA,采用ORF两端兼并引物,RT-PCR方法克隆梅花鹿天然Toll样受体9基因（TLR9）并进行系统的生物信息学分析。RT-PCR结果显示,梅花鹿TLR9基因的mRNA长4 043bp,含有800bp左右的5’UTR,完整ORF编码1 081个氨基酸（GenBank序列号为：HQ260632）。同源性分析显示梅花鹿TLR9基因编码区与其他物种高度同源,但5’UTR区存在基因结构的变异。功能结构域分析显示不同物种间TLR9结构域相对保守但也存在细微差别,结构域的差别导致梅花鹿TLR9蛋白胞外区马蹄形弧顶内侧空间结构由人类TLR9蛋白的弧形改变为梅花鹿TLR9蛋白的三角形。进化分析显示TLR9基因分子进化关系与物种间真实进化相一致。

Cloning and sequence analysis of Toll-like receptor 9 gene from sika deer

The total RNA from liver tissue was extracted by Trizol. The Toll-like receptor 9 gene （TLR9） was cloned by RT-PCR with degenerate primers,which was designed to locate On two ends of ORF. The deer TLR9 gene was analyzed by bio-informatics software. The result of RT-PCR showed the complete mRNA of deer TLR9 gene was 4 043 bp in length, containing a about 800 bp 5＇ UTR and a full ORF, coding 1 081 amino acids protein （GenBank Number：HQ260632）. Homology analysis showed that TLR9 gene has similar coding region to other mammal spe- cies,and has gene structure variation in 5 ＇ UTR region. The functional domain analysis showed that TLR9 proteins had high similarity in all species,but with a little of difference. The functional domain difference may caused tertiary structure change of deer TLR9 protein,in inside in top of arc of horseshoe-shape,in which was changed to from a cir- cular-arc in human TLR9 protein triangle in deer TLR9 protein. The evolution of TLR9 gene was consistent with species evolution. This study proved the deer TLR9 was conservative at sequence of coding region, functional domain and tertiary structure. This study will lay a foundation to understand deer TLR9 gene function.