草鱼TLR9基因全长cDNA的克隆及特征分析

Cloning and characterization of grass carp (Ctenopharyngodon idella)Toll-like receptor 9

采用同源克隆及快速扩增cDNA末端技术,从草鱼鳃组织中克隆了Toll样受体9(Toll-like receptor 9,TLR9)基因的cDNA全长。序列分析表明,草鱼TLR9 cDNA全长3 468 bp,编码1 058个氨基酸,其中包括18个氨基酸组成的信号肽、16个富含亮氨酸重复结构域(leucinerich repeat,LRR)、1个跨膜区和1个TIR结构域(Toll/IL-1 receptor)。该蛋白的分子量为121 921 u,等电点为8.80。氨基酸序列的同源性分析显示,草鱼TLR9与鲤TLR9的同源性最高(85%),依次为斑马鱼(82%)、大西洋鲑(55%)、虹鳟(55%)。在系统发生树上,草鱼TLR9首先与鲤科的鲤和斑马鱼聚类。通过半定量RT-PCR检测可知,草鱼TLR9在被检测的15个组织中都有表达,其中在鳃中的表达最高,其次为血、头肾等。这些结果为进一步深入研究TLR9的功能及开发草鱼免疫增强剂奠定基础。

The full-length cDNA sequence of Toll-like receptor 9（CiTLR9）gene was identified from grass carp（Ctenopharyngodon idella）gill by homologous cloning and rapid amplification cDNA ends（RACE）.The CiTLR9 cDNA was 3 468 bp,encoding 1 058 amino acid（aa）residues,including signal peptide,16 leucine rich repeat（LRR）motifs,one transmembrane zone and one Toll/IL-1 receptor（TIR）domain.The molecular weight of the deduced protein is 121 921 u,and the isoelectric point is 8.80.The amino acid sequence of CiTLR9 possessed 85%,82%,55%,and 55% identity with the TLR9s of Cyprinus carpio,Danio rerio,Salmo salar and Oncorhynchus mykiss respectively.CiTLR9 protein firstly clustered with TLR9s in Cyprinidae species,Cyprinus carpio and Danio rerio in the phylogenetic analysis.CiTLR9 mRNA was detected in all the tested 15 tissues（blood,brain,eye,foregut,midgut,hindgut,gas bladder,gill,head kidney,trunk kidney,heart,hepatopancreas,muscle,skin,spleen）by semi-quantitative real-time RT-PCR,highly in gill,blood and head kidney.The results serve further studies on functions of TLR9 and exploitation of immunostimulators in grass carp.