大菱鲆(Scophthalmus maximus L.)tlr3基因的鉴定及其在病原感染前后的表达

Identification,Characterization and Expression of tlr3 in the Mucosal Tissues of Turbot(Scophthalmus maximus L.)Following Bacterial Challenge

Toll样受体(Toll like receptor,TLR)是非常重要的病原体模式识别受体,它通过识别微生物特异性分子模式来控制针对病原体的宿主免疫应答,在先天免疫和获得性免疫中起重要作用。迄今为止,已经在动物中发现脊椎动物TLR的22个主要家族成员。尽管以前的研究已经鉴定了几个大菱鲆的Toll样受体基因(tlr2,tlr8,tlr9),但大菱鲆Toll样受体的深入研究可能会为检测大菱鲆适应性免疫应答反应提供有力依据。从之前的高通量测序数据库中可获得大菱鲆tlr3基因序列,但对其结构特征及其在黏膜免疫应答中的表达特征的研究依然欠缺。为更好地了解大菱鲆tlr3,本文鉴定其基因结构,预测蛋白质结构特征并进行系统进化分析,研究其在不同病原菌侵染黏膜屏障中的表达模式。结果表明:大菱鲆Tlr3包括13个亮氨酸富集的重复序列(LRR,leucine-rich repeats)和一个Toll样白介素-1受体(TIR),与牙鲆Tlr3同源性最高。进化树分析表明,大菱鲆Tlr3与牙鲆亲缘关系最接近。大菱鲆tlr3基因在健康组织中普遍表达,但是表达水平在8种检测组织中差异较大。大菱鲆tlr3在鳗弧菌和海豚链球菌侵染后的黏膜组织中均有表达上调的趋势。这些发现表明大菱鲆tlr3在受到包括革兰氏阴性菌和革兰氏阳性菌在内的广谱病原体感染的免疫应答中可能具有不可替代的作用。下一步可应用细菌学和免疫组织化学技术进一步研究黏膜组织上的主要细菌入侵位点,并确定侵染后大菱鲆TLRs的配体特异性。

Toll-like receptors(TLRs)are very important pathogen pattern recognition receptors.They control the host immune response against pathogens through recognition of molecular patterns specific to microorganisms,which plays an important role in innate immunity and acquired immunity.To date there are twenty-two major families of vertebrate TLRs have been found in animals.Although previous efforts have identified few TLRs(tlr2,tlr8 and tlr9)in turbot,a comprehensive investigation of the turbot TLRs could provide information for the detection of turbot adaptive immune response.Transcript of turbot tlr3 was captured from the previous high-throughput sequencing,however the research on its structural characteristics and its expression characteristics in mucosal immune response is still deficient.To better understand tlr3 in turbot,we predicted its protein structure,analyzed its phylogenetic relationship with other species and characterized its expression patterns with emphasis on mucosal barriers following different bacterial infections.The results showed that turbot Tlr3 included 13 leucine-rich repeats(LRR)and a Toll-like interleukin-1 receptor(TIR),which had the highest homology with flounder Tlr3.The phylogenetic analysis revealed that the turbot toll-like receptor 3 gene showed the closest relationship to Paralichthys olivaceus.The tlr3 gene was ubiquitously expressed in healthy tissues although expression levels varied among the eight tested tissues.Turbot tlr3 had a tendency of up-regulation in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenges infected by Vibrio anguillarum and Streptococcus iniae.These findings suggest a possible irreplaceable role of TLRs in the immune responses to the infections of a broad range of pathogens that include Gram-negative and Gram-positive bacteria.Future studies should apply the bacteriological and immune-histochemical techniques to study the main sites on the mucosal tissue for bacterial entry and identify the ligand specificity of the turbot TLRs after challenge.