| Large yellow croaker(Larimichthys crocea)as a high production Marine economic fish,its economic value is particularly important in seafood in China.At present,the cultuerd technology rapid development and aquaculture production improve quickly,the disease is the most serious problem in the process of large yellow croaker cultured,which restrict the development of large yellow croaker breeding.Therefore,enhancing the basic research of large yellow croaker immunology theory and understanding theory of the immune mechanism are very important to the large yellow croaker for disease control and prevention.And the development of aquaculture can continuous improvement.This paper analyzes the genetic characteristics and molecular structures of Rab5 A,Rab11 and Rab13 in large yellow croaker,which belong with small GTP protein family.The transcription and expression of Rab5 A,Rab11 and Rab13 were researched by RT-PCR in different tissues of large yellow croaker.To detect the change of expression in liver,spleen and kidney,after stimulating large yellow croaker with Cryptocaryon irritans brown,Vibrio parahaemolyticus,LPS,poly I: C,then recombined and expressed the Rab5 A in Escherichia coli and purified.Subsequently,the specific antibody was raised using the purified fusion protein.Moreover,the titre and the optimal working concentration of the Rab5 A antibody were detected.Western blot experiment researched the Rab5 A protein expression in tissues and immune stimulation expression changes in the organizations.The Rab5 A interacting proteins were analyse by the GST pull-down.Immune electron microscopy(sem)experiments detected the subcellular localization of Rab5 A and Rab13 in different organization.The results are as follows:(1)The large yellow croaker Rab5 A gene contains a 1 092 bp cDNA and a 651 bp ORF,encoding 216 amino acids.The coding protein structure contained a typical RAB function structure domain.The protein molecular weight was about 23.56 KDa.The Real-time PCR analysis found that Rab5 A is widely expressed in skin,liver,heart,gill,kidney,intestine,spleen,blood and stomach tissues of large yellow croaker.And the highest expression quantity was blood.Cryptocaryon irritans hatched in 2 hours were used to infect large yellow croaker.At 6 h,24 h,48 h,and 72 h;the liver,spleen and kidney were collected.The results showed that Rab5 A expression levels were significantly up-regulated in liver,spleen and kidney(P<0.05).And they all reached their highest value at 24 h.Based on the protein interaction studies.The GST pull-down assay showed that Rab5 A interacted with Myosin light chain,lymphatic tyrosine kinase and Leptin.By intraperitoneal injection of PBS,LPS,poly real I: C and deputy hemolytic vibrio with large yellow croaker,the samples were collected in 3 h-72 h.Real-time PCR and Western blot detected the expression of Rab5 A before and after immune situation in liver,spleen and kidney.Results showed that the large yellow croaker Rab5 A gene expression level increases obviously in the liver,spleen and kidney.Immune electron microscopy detected the subcellular localization of Rab5 A.The results showed that Rab5 A localization in the nucleus and mitochondria at the brain and in the fat granule at the liver.(2)The full length of Rab11 cDNA was 1 373 bp,including ORF of 657 bp,encoding 218 amino acids.The coding protein structure contained a typical RAB function structure domain.The protein molecular weight was about 24.45 KDa.The predicted phosphorylation site analysis showed that there are six serine phosphorylation sites;one threonine phosphorylation site and two tyrosine phosphorylation sites in Rab11.The Real-time PCR analysis found that Rab11 is widely expressed in nine tissues of large yellow croaker.And the highest expression quantity was blood.Cryptocaryon irritans hatched in 2 hours were used to infect large yellow croaker.The results showed that Rab11 expression levels were significantly up-regulated in liver,spleen and kidney(P<0.05).And they all reached their highest value at 24 h?72 h and 48 h.After intraperitoneal injection of PBS,LPS,poly I:C and deputy hemolytic vibrio with large yellow croaker.Real-time PCR detected the expression of Rab11 before and after immune situation in liver,spleen and kidney.Results showed that gene expression level of Rab11 increases obviously in the liver,spleen and kidney.(3)The full length of Rab13 cDNA was 2 390 bp,including ORF of 603 bp,encoding 200 amino acids.The protein molecular weight was about 22.4 KDa.The Real-time PCR analysis found that Rab13 is widely expressed in 9 tissues of large yellow croaker.The highest expression is in kidney,expressed in gill and liver volume is higher,The lowest expression is in stomach.Cryptocaryon irritans hatched in 2 hours were used to infect large yellow croaker.The gene expression changes of Rab13 was similar as Rab5 A.The results showed that Rab11 expression levels were significantly up-regulated in liver,spleen and kidney(P<0.05).And they all reached its highest value at 48 h.By intraperitoneal injection of PBS,LPS,poly real I: C and deputy hemolytic vibrio with large yellow croaker,the samples were collected in 3 h-72 h.Real-time PCR detected the expression of Rab13 before and after immune situation in liver,spleen and kidney.Results showed that the Rab13 gene expression level increases obviously in these tissues.Detection of Rab11 subcellular localization with immune electron microscopy(sem)showed that Rab13 localization in the cell membrane on the surface of the fiber in the spleen.The researches showed that Rab13 plays an important role in transport regulation of intercellular substance.The investigation showed that Rab5 A,Rab11 and Rab13 genes might play an important role in defensing against parasite,bacteria and virus in large yellow croaker immune. |
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