渔业科学进展2018，Vol.39Issue(3)：53-64,12.DOI:10.19663/j.issn2095-9869.20170410001
牙鲆NOD2基因的表达分析及 在抗迟缓爱德华氏菌感染过程中的功能
Expression Analysis and Functional Characterization of NOD2 in the Resistance of Japanese Flounder (Paralichthys olivaceus) to Edwardsiella tarda Infection
摘要
Abstract
The innate immune system acts as the first defense against invading pathogens through three classes of pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs), including the Toll-like receptors (TLRs), RIG-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). The nucleotide-binding oligomerization domain-like receptors are a family of innate immune receptors that link cytosolic sensing of microbes and danger stimuli to activate immune responses. As a member of the NOD-like receptor family, NOD2 can recognize bacterial peptidoglycan and activate immune responses via nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). In the present study, the Japanese flounder (Paralichthys olivaceus) NOD2 (PoNOD2) was identified by searching the transcriptome sequence of Japanese flounder based on the available NOD2 cDNA sequences of other fishes. The complete open reading frame (ORF) of PoNOD2 is 2964 bp and encodes a polypeptide of 988 amino acids. The gene PoNOD2 is composed of three main domains: an N-terminal domain containing two adjacent CARDs, a central NACHT domain, and a multiple C-terminal LRRs. The phylogenetic tree analysis suggests that the gene PoNOD2 is closely related to that of the Fugu rubripes (Takifugu rubripes). Tissue expression analysis by qRT-PCR showed that the gene PoNOD2 is constitutively expressed in all the examined tissues, predominantly in the spleen and liver. Real-time PCR showed that the infection of Edwardsiella tarda can significantly upregulate the expression of PoNOD2 in the tissue of Japanese flounder. In vitro immune stimulation experiments showed that PoNOD2 expression could be upregulated by peptidoglycan, polyinosinic: polycytidylic acid, and E. tarda infection. Subcellular localization of the PoNOD2 protein is in the cytoplasm of the gill cells of flounder. When the flounder gill (FG) cells were infected by E. tarda, the overexpression of PoNOD2 can inhibit the bacterial replication and upregulate the transcription of pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-8. These results suggest that PoNOD2 plays a key role in the resistance of P. olivaceus to E. tarda infection and in the modulation of immune responses.关键词
牙鲆/模式识别受体/NOD2/迟缓爱德华氏菌/牙鲆鳃细胞系Key words
Japanese flounder (Paralichthys olivaceus)/PRR/NOD2/Edwardsiella tarda/FG cells分类
生物科学引用本文复制引用
曹丹丹,刘金相,王志刚,张全启,齐洁,王旭波,于海洋..牙鲆NOD2基因的表达分析及 在抗迟缓爱德华氏菌感染过程中的功能[J].渔业科学进展,2018,39(3):53-64,12.基金项目
国家自然科学基金(31101891)和国家 863 高技术计划(2012AA10A408)共同资助 [This work was supported by the National Natural Science Foundation of China (31101891), and the National High Technology Development Program of China (863) (2012AA10A408)]. （31101891）
