Molecular characterization of Fyn, Lck and Zap-70 in golden pompano (Trachinotus ovatus)
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Youchuan Wei1, Dongqiang Gan1, Wenrui Qi2
1College of Animal Science and Technology, Guangxi University, Nanning, Guangxi Zhuang Autonomous Region, 530005, China.
Abstract:
T-cell receptor (TCR) signaling is essential for adaptive immunity in vertebrates, with the tyrosine kinases Fyn, Lck, and Zap-70 playing central roles in signal transduction. In the present study, the full cDNA sequences of Fyn, Lck, and Zap-70 from golden pompano (Trachinotus ovatus) were obtained by RT-PCR and RACE-PCR and their expression patterns in normal tissues and head kidney (HK) following lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (polyI:C) stimulation and Vibrio alginolyticus infection were examined by qPCR. The open reading frames of trFyn, trLck, and trZap-70 respectively encoded 537, 513, and 609 amino acids, all containing conserved similar functional domains as their mammalian counterparts. qPCR analysis revealed that all three genes were ubiquitously expressed in examined tissues, with high expression in immune-related tissues. trFyn, trLck, and trZap-70 were significantly induced in HK after LPS, polyI:C stimulation and V. alginolyticus infection. Co-immunoprecipitation (co-IP) assay demonstrated that both trFyn and trLck could interact with trZap-70 in HEK-293T cells. These findings provide molecular basis for further study of the TCR signaling and T-cell mediated immunity in golden pompano.
Insights
This study characterizes the Fyn, Lck, and Zap-70 genes in golden pompano, revealing their crucial roles in T-cell receptor signaling and immune responses. These findings enhance our understanding of adaptive immunity in fish.
Area of Science:
- Immunology
- Molecular Biology
- Aquatic Animal Health
Background:
- T-cell receptor (TCR) signaling is vital for adaptive immunity in vertebrates.
- Tyrosine kinases Fyn, Lck, and Zap-70 are key players in TCR signal transduction.
- Understanding these pathways in fish is crucial for aquaculture and comparative immunology.
Purpose of the Study:
- To isolate and characterize the cDNA sequences of Fyn, Lck, and Zap-70 in golden pompano (Trachinotus ovatus).
- To investigate the expression patterns of these genes in various tissues and following immune stimulation.
- To explore the protein interactions between Fyn, Lck, and Zap-70 in this species.
Main Methods:
- Rapid amplification of cDNA ends (RACE-PCR) and reverse transcription PCR (RT-PCR) for gene isolation.
- Quantitative PCR (qPCR) to analyze gene expression levels.
- Co-immunoprecipitation (co-IP) assays to determine protein interactions.
Main Results:
- Full cDNA sequences for golden pompano Fyn, Lck, and Zap-70 (trFyn, trLck, trZap-70) were obtained, encoding proteins with conserved functional domains.
- These genes were ubiquitously expressed, with higher levels in immune-related tissues.
- Expression of trFyn, trLck, and trZap-70 was significantly induced in head kidney following stimulation with LPS, polyI:C, and Vibrio alginolyticus infection.
- trFyn and trLck were shown to interact with trZap-70.
Conclusions:
- The characterized golden pompano Fyn, Lck, and Zap-70 genes are involved in T-cell receptor signaling.
- Their induction upon immune challenge highlights their role in adaptive immunity in fish.
- These findings lay the groundwork for further research into T-cell mediated immunity in golden pompano.
