Molecular Characterizations And Functions Of Ran,Rab, Rac In Large Yellow Croaker

Large yellow croaker (Larimichthys crocea Richardso,1846) is an economically important mari-cultured fish species in China. However, in recent years, with the rapid development of the large yellow croaker culture industry, the infectious diseases caused by viruses and bacteria, are becoming more and more severe, resulting in great economic losses. Therefore, there is an urgent need to understand the immune response of large yellow croaker to control the diseases and for long-term sustainability.In the present investigation, the full-length cDNA of Ran, Rab, Rac and the factors interacted with them were cloned by RT-PCR and RACE-PCR techniques in large yellow croaker. Adiditionally, the tissue expression and temporal expression profiles of these genes after stimulation with formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus, polyinosinic polycytidynic acid (Poly I:C), and bacterial lipopolysaccharides (LPS). It was found these genes were ubiquitously transcribed and expressed in9tissues although the levels were different among thess tissues. Time couse analysis revealed that their expression were obviously up-regulated in blood, spleen and liver after immunization. Furthermore, the proteins interacted with Ran, Rab and Rac has been studied by GST-pull down assay. The article has got the main results as follows:1. The full-length cDNA of LycRan was of1033bp, including an open reading frame (ORF) of648bp encoding a polypeptide of215amino acids. The GTP-binding assay showed that the Ran protein had GTP-binding activity. The results of RT-PCR and Western blot indicated that the transcript and protein of Ran were detected in every tissue of large yellow croaker including kidney, liver, gill, muscle, spleen, skin, heart, intestine and blood. The result of quantitative Real-Time RT-PCR analysis revealed the highest expression in kidney and the weakest expression in skin. Time couse analysis showed that LycRan expression was obviously up-regulated in kidney, blood and spleen after immunization with either Poly I:C or formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus. Based on protein interactions, it was found that the LycRan was interacted with myosin light chain (MLC) protein, a crucial protein in the process of phagocytosis to form a protein complex.2. The full-length cDNA of LycRab was of796bp, including an open reading frame (ORF) of624bp encoding a polypeptide of207amino acids. The GTP-binding assay showed that the Rab protein had GTP-binding activity. The results of RT-PCR and Western blot indicated that the transcript and protein of Rab were detected in the9tissues. The result of quantitative Real-Time RT-PCR analysis revealed the highest expression in blood and the weakest expression in skin. Time couse analysis showed that LycRab expression was obviously up-regulated in kidney, btood and spleen after immunization with either virus or bacteria. Based on protein interactions, it was found that the LycRab was interacted with Actin.3. The full-length cDNA of LycRac was of1221bp, including an open reading frame (ORF) of579bp encoding a polypeptide of192amino acids. The GTP-binding assay showed that the Rac protein had GTP-binding activity. The results of RT-PCR and Western blot indicated that the transcript and protein of Rac were detected in the9tissues. The result of quantitative Real-Time RT-PCR analysis revealed the highest expression in gill and the weakest expression in spleen. Time couse analysis showed that LycRac expression was obviously up-regulated in kidney, blood and spleen after immunization with either virus or bacteria. Based on protein interactions, it was found that the LycRac was interacted with tropomyosin (TPM).These results suggested that Ram Rab and Rac protein play important roles in the immune response against pathgons in large yellow croaker. These information could contribute better understanding to the still poorly understood molecular events involved in fish immune response. MLC, Actin and TPM were crucial proteins in the process of phagocytosis involved innate immune. These results suggested that Ran, Rab and Rac protein play important roles in the immune response against pathgons in large yellow croaker. These information could contribute better understanding to the still poorly understood molecular events involved in fish immune response.