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[目的]克隆牙鲆TLR1(Toll like receptors)全长基因,并对其结构特征和表达规律进行分析。[方法]采用同源克隆和快速扩增cDNA末端技术,从牙鲆头肾组织中克隆出TLR1基因cDNA全长序列,并对该基因进行生物信息学和表达模式分析。[结果]牙鲆TLR1基因cDNA全长2947bp,开放阅读框(ORF)2418bp,编码805个氨基酸,包括26个氨基酸组成的信号肽、2个跨膜区、6个富含亮氨酸重复结构域(LRR)和一个TIR结构域(Toll/interleukin(IL)-1receptor)。该蛋白的分子量为91.15kDa,等电点为6.49。氨基酸序列同源性分析显示,牙鲆TLR1基因与其他脊椎动物的TLR1基因序列全长同源性达到69%~35%,TIR序列的同源性达到84%~62%。在系统发生树上牙鲆的TLR1基因首先与斜带石斑鱼聚类。通过荧光定量qRT-PCR检测,结果显示牙鲆TLR1基因的mRNA主要表达于肝脏、心脏和脾脏等组织。[结论]该研究结果为进一步研究TLR1基因的功能和开发牙鲆免疫增强剂奠定了基础。
[Objective] To clone full-length TLR1 (TLR1) gene and analyze its structure and expression pattern. [Method] The full-length cDNA sequence of TLR1 gene was cloned from the head and neck of Japanese flounder by homologous cloning and rapid amplification of cDNA ends. The bioinformatics and expression patterns of TLR1 gene were analyzed. [Result] The TLR1 gene cDNA of Paralichthys olivaceus was 2947bp in length and 2418bp in open reading frame (ORF). It encoded 805 amino acids including 26 amino acid signal peptide, two transmembrane domains and six leucine - rich repeat domains (LRR) and a TIR domain (Toll / interleukin (IL) -1 receptor). The protein has a molecular weight of 91.15 kDa and an isoelectric point of 6.49. Homology analysis of amino acid sequence showed that the TLR1 gene of Japanese flounder shared 69% -35% homology with TLR1 gene sequences of other vertebrates and the homology of TIR sequences was 84% -62%. In the phylogenetic tree, the TLR1 gene from Japanese flounder is clustered first with the grouper. The result of qRT-PCR showed that the TLR1 mRNA of Japanese flounder was mainly expressed in liver, heart and spleen. [Conclusion] The results of this study lay the foundation for further study on the function of TLR1 gene and the development of immune enhancer of Japanese flounder.