HcTLR1通过MyD88-NF-κB信号通路参与三角帆蚌抗菌免疫应答  

HcTLR1 involved in antimicrobial immune response by MyD88-NF-κB signaling pathway in Hyriopsis cumingii

作　　者：路俊怡 姜立妍 龙凯 王韬 吴正理[1] 李艳红[1] LU Junyi;JIANG Liyan;LONG Kai;WANG Tao;WU Zhengli;LI Yanhong(College of Fisheries,Southwest University/Research Center for Aquatic Biodiversity Conservation in the Upper Reaches of Yangtze River,Chongqing 400715,China)

机构地区：[1]西南大学水产学院/农业农村部长江上游水生生物多样性保护研究中心,重庆400715

出　　处：《南方水产科学》2024年第6期19-30,共12页South China Fisheries Science

基　　金：重庆市自然科学基金面上项目(cstc2021jcyj-msxmX1202);重庆市生态渔业产业技术体系(CQMAITS202315);重庆市水产科技重点攻关项目(CQFTIU2024-09)。

摘　　要：Toll样受体(Toll-like receptors,TLR)家族是一类进化保守的病原体识别受体,在检测和防御微生物病原体的先天免疫中发挥着重要作用。为了探究HcTLR1基因在三角帆蚌(Hyriopsis cumingii)抗菌应答中的作用,使用cDNA末端快速克隆技术(Rapid-amplification of cDNA ends,RACE)获得HcTLR1基因cDNA全长序列,采用实时荧光定量PCR分析比较HcTLR1基因在三角帆蚌不同组织和不同免疫刺激后的表达水平,利用双链RNA干扰技术分析敲低该基因后MyD88依赖性通路及相关免疫基因的变化。结果显示,HcTLR1基因开放阅读框全长为3687 bp,编码1228个氨基酸。预测的HcTLR1蛋白结构包含多个亮氨酸富集的重复序列(Leucine-rich repeat,LRR)结构域、1个跨膜结构域和1个胞内Toll/白细胞介素-1(Toll-IL-1 receptor domain,TIR)受体。此外,HcTLR1基因在血细胞中表达量最高,且对维氏气单胞菌GL1(Aeromonas veronii GL1)和病原体相关分子模式(Pathogen-associated molecular patterns,PAMPs)刺激呈现出差异显著的时间依赖性变化。敲低HcTLR1基因后,维氏气单胞菌GL1刺激所激活的MyD88相关通路基因、抗菌肽、溶菌酶、防御素、乳清酸性蛋白、脂多糖结合蛋白/杀菌通透性增加蛋白2和白细胞介素17等基因的表达水平显著降低。结果表明,HcTLR1参与了三角帆蚌在微生物感染过程中MyD88依赖性信号通路的活化,并促进了血细胞抵抗机制。Toll-like receptor(TLR)family is an evolutionarily conserved pathogen recognition receptor,playing an important role in detecting and defending against microbial pathogens.To study the role of HcTLR1 gene in the antimicrobial response of Hyriopsis cumingii,the full-length cDNA sequence of HcTLR1 gene was cloned using rapid-amplification of cDNA ends(RACE)technology;real-time fluorescence quantitative PCR analysis was employed to compare the expression levels of HcTLR1 gene in various tissues of H.cumingii challenged with different stimuli;double-stranded RNA interference technology was used to analyze the changes in MyD88-dependent pathway and related immune genes after the knockdown of the gene.The results show that the open reading frame(ORF)of HcTLR1 gene was 3687 bp,encoding 1228 amino acids.The predicted structure of HcTLR1 protein contained multiple Leucine-rich repeat domains,a transmembrane domain and an intracellular Toll/interleu-kin-1 receptor.Furthermore,the mRNA expression of HcTLR1 gene was highest in hemocytes and exhibited significant changes in response to Aeromonas veronii GL1 and pathogen-associated molecular patterns stimulation at different time points.Moreover,the knockdown of HcTLR1 gene significantly reduced the expression levels of genes in MyD88-related pathway,anti-bacterial peptides,lysozyme,defensins,lactoferrin,LPS1-binding protein/bactericidal permeability-increasing protein 2,and in-terleukin 17 stimulated by A.veronii GL1.In conclusion,it is suggested that HcTLR1 activates MyD88-dependent signaling pathways in H.cumingii during microbial infection and promotes resistance mechanisms in hemocytes.

关 键 词：三角帆蚌 HcTLR1基因 先天性免疫 RNA干扰 

分 类 号：S917.4[农业科学—水产科学]