牙鲆Toll样受体1基因全长cDNA的克隆及特征分析(英文)

Molecular Cloning and Expression Analysis of Toll-like Receptor 1 cDNA in Japanese Flounder,Paralichthys olivaceus

[目的]克隆牙鲆TLR1（Tolllikereceptors）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TLRl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K2947bp，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TLRl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] The paper aimed to clone the full length gene of Toll-like recep- tors （TLRs） in Japanese flounder （Paralichthys olivaceus）, and analyze their structural features and expression regularity. [Method] The full length cDNA sequence of Toll like receptor 1（TLR1） gene was identified from Japanese flounder head kidney by ho- mologous cloning and rapid amplification cDNA ends （RACE）. The bioinformatics and expression model of this gene was analyzed. [Result] The TLR1 cDNA was 2 947 bp, a 2 418 bp open reading frame （ORF）, encoding 805 amino acid （aa） residues, including signal peptide, six leucine-rich repeat（LRR） motifs, two transmembrane zones and one TolI/IL 1 receptor （TIR） domain. The molecular weight of the deduced protein was 91.15 KDa, and the isoelectric point was 6.49. The amino acid sequence of Japanese flounder TLR1 possessed 69%-35% identity with the TLRls of other verte- brates, further analysis showed that the TIR domain of Japanese flounder TLR1 shared 84%-62% identities with TIR domains in other vertebrates. Japanese flounder TLR1 protein firstly clustered with TLRls in Epinephelus coioides in the phylogenetic analysis. The transcription of Japanese flounder TLR1 was examined by real-time quantitative PCR, and its mRNA was mainly detected in liver, heart and spleen. [Conclusion] The results lay a foundation for further studying the functions of TLR1 and developing immune potentiator in Japanese flounder.