Abstract:
【Objective】To investigate the structural characteristics,tissue expression distribution of
BAG-1 gene (
EcBAG-1)in
Epinephelu coioides and its expression changes of it after stimulation of lipopolysaccharide(LPS)and polyinosinic acid(PolyI∶C),with an aim to further reveal the mechanism of fish BAG-1 in pathogen infection,so as to provide a reference for understanding the immunity of fish against disease.【Method】The
EcBAG-1 gene was cloned by designing primers based on its EST sequence,and bioinformatics analysis was performed by using online software such as SMART,TMHMM Server v.2.0,SOPMA,SWISS-MODEL,ExPASy,ClustalX and MEGA 5.0. The tissue expression characteristics of
EcBAG-1 gene and its expression pattern after LPS and Poly I:C stimulation were detected by using realtime fluorescence quantitative PCR.【Result】The open reading frame(ORF)of the cloned
EcBAG-1 gene was 624 bp in length,encoding 207 amino acids,and EcBAG-1 protein molecular weight was 49.84 kD,theoretical isoelectric point (pI)was 5.18 and it was a hydrophobic protein. Ec-BAG-1 protein had a conserved BAG domain and an UBQ ubiquitinrelated domain of the BAG-1 family,and no transmembrane domain,it mainly existed in the cytoplasm and nucleus. In the secondary structure,α - helix accounted for 65.22%,extended chain 10.63%,irregular coiled coil 20.29% and β - turned angle only 3.86%. EcBAG-1 amino acid sequence of
E. coioides had the highest similarity(96.62%)with
Larimichthys crocea,and the two species was the most closely related.
E. coioides was relatively distantly related to
Gallus gallus and
Xenopus tropicalis,with corresponding BAG-1 amino acid sequence similarities of 58.45% and 57.50%,respectively.
EcBAG-1 gene was expressed in liver,thymus,spleen,head kidney,gill,skin,muscle,brain,peripheral blood lymphocytes and heart of
E. coioides,and the relative expression was highest in brain,which was significantly higher than that in other tissues(
P>0.05,the same below). After 24 h of LPS and PolyI:C stimulation,relative expression of
EcBAG-1 gene in the spleen of
E. coioides.【Conclusion】EcBAG-1 protein contains the typical BAG structural domain and UBQ structural domain of the BAG-1 protein family,and it has biological function similar to mammalian BAG-1 of mammals,which means
EcBAG-1 gene plays an important role in the resistance to bacterial/viral infection in
E. coioides, broadening the idea of understanding the anti-infection mechanism in fish.