卵形鲳鲹TLR13基因的克隆、表达分析和亚细胞定位研究

Cloning, expression analysis, and subcellular localization of the TLR13 gene of golden pompano (Trachinotus ovatus)

中文关键词: 形鲳鲹  TLR13  基因克隆  表达谱  亚细胞定位

英文关键词: Trachinotus ovatus  TLR13  Gene cloning  Expression profile  Subcellular localization

中文摘要:

      Toll样受体(Toll-like receptor, TLR)是一种古老的先天性免疫受体，参与病原体相关分子模式识别，对维持免疫稳态和预防感染至关重要。本研究克隆和鉴定了卵形鲳鲹(Trachinotus ovatus) TLR13基因(命名为ToTLR13)，其开放阅读框(ORF)为1 269 bp，编码422个氨基酸，等电点为8.13。保守结构域分析显示，ToTLR13含有跨膜结构域(TM)、LRR结构域和TIR结构域，符合TLR家族的典型特征。通过建立TLR13保守域三级结构发现，ToTLR13与小鼠(Mus musculus)和大黄鱼(Larimichthys crocea) TLR13功能结构域的蛋白三级结构具有较高重叠性。多序列比对显示，ToTLR13与其他硬骨鱼TLR13具有较高的相似性，与其他纲物种的序列相似性较低。系统进化树结果显示，ToTLR13与硬骨鱼TLR13聚在一起，其中与鞍带石斑鱼(Epinephelus lanceolatus)最为接近，与哺乳动物、两栖类和贝类相分离。实时荧光定量PCR (Real-time fluorescence quantitative PCR, RT-qPCR)分析显示，ToTLR13在健康卵形鲳鲹的心、鳃、肾、头肾、肝、脾、脑和肌肉中普遍表达，其中鳃的表达量最高，其次是脾。ToTLR13在其鳃、脾、肝和肾免疫相关组织中的表达情况呈现出不同程度的上调，提示其经无乳链球菌(Streptococcus agalactiae)和溶藻弧菌(Vibrio alginolyticus)免疫刺激后可能激活了炎症反应，启动了先天性免疫反应。亚细胞定位显示，ToTLR13定位于A549细胞质。本研究表明，ToTLR13在抵御病原菌免疫应答过程中可能发挥重要的作用，研究结果可为阐明脊椎动物TLRs的功能进化史提供基础资料。

英文摘要:

      The innate immune response serves as the first line of defense and is initiated through the sensing of pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs) are ancient innate immune receptors involved in pathogen-related molecular pattern recognition, which is essential for immune homeostasis and the prevention of infection. As a member of the TLR11 family, TLR13 has been identified in several teleost fishes, including Larimichthys crocea, Oreochromis niloticus, and Epinephelus coioides. These studies have mainly focused on the function of TLR13 in protecting the body from bacterial or viral invasion. To further investigate the immune function of TLR13, the gene of open reading frame (ORF) sequence of TLR13 (ToTLR13) from golden pompano (Trachinotus ovatus) was cloned and characterized in this study. The expression pattern of ToTLR13 was determined in healthy tissues and infected immune-related tissues in golden pompano by real-time fluorescence quantitative PCR (RT-qPCR). Moreover, subcellular localization of ToTLR13 in A549 cells was determined. The results showed that the ORF sequence of ToTLR13 was 1 269 bp, encoding 422 amino acids with an isoelectric point of 8.13. ToTLR13 was classified as a hydrophilic protein by hydrophilic prediction. In addition, ToTLR13 contains a 15-amino-acid-coded signal peptide. Conservative structure domain analysis showed that ToTLR13 contains a transmembrane (TM) domain, a leucine-rich repeat (LRR) domain involved in ligand recognition and binding, and a conserved Toll/interleukin-1 receptor (TIR) domain involved in signal transduction, which is in line with the typical characteristics of the TLR family. The TIR domain exists in almost all transmembrane TLRs and its sequence is highly conserved. By establishing the tertiary structure of the conserved domain of TLR13, ToTLR13 has a high spatial structure that overlaps with the LRR and TIR domains of Mus musculus and L. crocea TLR13, which shows that the TLR13 structure and function in different species have a certain similarity. Multiple sequence alignment showed that ToTLR13 had a high similarity with other teleost fish TLR13 (82.52%~84.58%), while with other classes of species, sequence similarity was low (33.30%~46.11%). Furthermore, according to the phylogenetic tree analysis, we found that the relationship between ToTLR13 and other teleost fish TLR13 is relatively close, among which Epinephelus lanceolatus is the closest evolutionary position. While it is distant from other species, mammals are grouped into one branch; Xenopus tropicalis and Cyclina sinensis are in another branch. RT-qPCR results revealed that ToTLR13 was constitutively expressed, with the highest expression level in the gill and spleen, followed by the brain, liver, and kidney, and expression was lower in the heart, head kidney, and muscle. The mRNA expression of TLR13 is slightly different in different fish, which indicates that TLR13 has species specificity and tissue specificity in normal fish tissues. Moreover, TLR13 is generally highly expressed in fish immune-related tissues, suggesting that TLR13 may play different roles in different fish species and plays an important immunomodulatory role. When stimulated by pathogens or viruses, the mRNA expression of TLR13 in immune-related tissues of different fish varies. In this study, after infection with Streptococcus agalactiae and Vibrio alginolyticus, there were significant changes in the mRNA expression of ToTLR13 in different tissues. The ToTLR13 mRNA expression level in the gill suddenly reached a peak at 72 h after infection with S. agalactiae, but showed significant differences at 12 h and 96 h in the V. alginolyticus experimental group. In the spleen, the mRNA expression of ToTLR13 increased in a time-dependent manner after infection with S. agalactiae and V. alginolyticus, peaking at 24 h and 96 h, respectively. The mRNA expression level of ToTLR13 in the liver showed a regular trend of increasing and then decreasing from 0 h to 48 h after S. agalactiae infection and reached a peak at 72 h. In the V. alginolyticus experimental group, the mRNA expression level of ToTLR13 in the liver decreased to below the original level at first and then increased, reaching a peak at 48 h. In the kidney, the ToTLR13 mRNA expression level in the S. agalactiae group reached a double peak at 12 h and 72 h after infection, respectively. ToTLR13 mRNA expression level reached a peak at 6 h after V. alginolyticus infection, and then decreased to the level before challenge. These results suggest that ToTLR13 plays an important role in the immune response against pathogenic bacteria. According to their intracellular localization, TLRs can be divided into two categories: Those expressed on the surface of the cell membrane and those localized in the cytoplasm. In this study, subcellular localization showed that ToTLR13 was localized in the cytoplasm of A549 cells, and this phenomenon was also found in other teleost fish TLR13. The results of this study showed that ToTLR13 might be involved in the innate immunity of golden pompano against pathogenic bacteria, which lays a foundation for studies on the function of TLR13 in teleost.