Characterization of myosin light chain gene up-regulated in the large yellow croaker immunity by interaction with RanGTPase.

RanGTPases are highly conserved in eukaryotes from yeast to human and have been implicated in many aspects of nuclear structure and function. In our previous study, it was revealed that the RanGTPase was up-regulated in large yellow croaker challenged by pathogen. However, the mechanism of RanGTPase in immunity remains unclear. In this investigation, on the basis of protein interaction, it was found that RanGTPase interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. Furthermore, it was found and characterized in this marine fish for the first time. The full-length cDNA of LycMLC was 771bp, including a 5'-terminal untranslated region (UTR) of 36bp, 3'-terminal UTR of 279bp and an open reading frame (ORF) of 456bp encoding a polypeptide of 151 amino acids. RT-PCR analysis indicated that LycMLC gene was constitutively expressed in the 9 tissues examined, including kidney, liver, gill, muscle, spleen, skin, heart, intestine and blood. The result of quantitative real-time PCR analysis revealed the highest expression in muscle and the weakest expression in skin. Time course analysis showed that LycMLC expression was obviously up-regulated in blood after immunization with either poly I:C or formalin-inactive Gram-negative bacteria Vibrio parahaemolyticus. It indicated that the highest expression was 4.5 times (at 24h) as much as that in the control (P<0.05) challenged by poly I:C and 5.0 times (at 24h) challenged by bacteria. These results suggested that LycMLC might play an important role in large yellow croaker defense against the pathogen infection. Therefore our study revealed a novel pathway concerning immunity of RanGTPase by the direct interaction with the cytoskeleton protein, which would help to better understand the molecular events in immune response against pathogen infection in fish.