Study On The Immune Function Of Antimicrobial Peptides(AMPs) And Lectin Genes From Yellow Drum Nibea Albiflora

Antimicrobial peptides and lectins are non-specific immune factors in fish,they are not only the first line of defense against pathogen infection,but also can activate specific immune responses.In this study,three lectin genes and two antimicrobial peptide genes were identified from the transcriptome database of Nibea albiflora,named YdGal-3,YdGal-9,YdGal-1,YdNkl-1and YdNkl-2,respectively.Meanwhile,the studies on their genetic characteristics and immune function have been conducted.The results obtained are as follows:YdGal-3,YdGal-9,and YdGal-1 belong to the galectin family,without signal peptide and transmembrane region,and have conserved sugar binding motifs and carbohydrate recognition domains（CRD）.The YdGal-3 encoding 383 amino acids,YdGal-9 encoding 349 amino acids,and YdGal-1 encoding 135 amino acids.The results of q RT-PCR showed that the m RNAs of YdGal-3,YdGal-9,and YdGal-1 were widely expressed in 12 tissues of N.albiflora,YdGal-3had the highest expression in the brain;YdGal-9 had a higher expression level in the kidney,head-kidney,and liver;and YdGal-1 had a higher expression level in the swim bladder and gill.After being infected by Vibrio harveyi,the expression levels of YdGal-3 in the head-kidney,liver,and spleen were significantly increased;the expression levels of YdGal-9 and YdGal-1 in the brain were significantly increased.The subcellular localization results showed that YdGal-3,YdGal-9,and YdGal-1 protein were all distributed in the cytoplasm and nucleus in HEK 293T cell.The purified YdGal-3,YdGal-9,and YdGal-1 protein could agglutinated the red blood cells of rabbit,large yellow croaker,and N.albiflora without the action of Ca²⁺.In addition,they can agglutinated some gram-negative bacterias and exhibit antibacterial activity.In addition,YdGal-3 can agglutinated four gram-negative bacterias including Pseudomonas plecoglossicida,Vibrio parahaemolyticus,V.harveyi,and Aeromonas helphia;YdGal-9 can agglutinated six gram-negative bacterias including P.plecoglossicida,V.parahaemolyticus,V.harveyi,A.helphia,Escherichia coli,and Vibrio alginolyticus;YdGal-1 can agglutinated six gram-negative bacterias including Edwardsiella tarda,E.coli,Photobacterium phosphoreum,A.helphia,Pseudomonas aeruginosa,and Aeromonas veronii.It was also found by scanning electron microscope that YdGal-3 could damaged the cell membrane of P.plecoglossicida and V.parahaemolyticus.Sugars including lactose,D-galactose and lipopolysaccharide（LPS）could inhibited the aggregation activity of YdGal-3,YdGal-9,and YdGal-1 protein.Furthermore,the aggregation mechanism of YdGal-3 and YdGal-9 was further studied by deleting the sugar binding motifs,and the results showed that different sugar binding motifs can bind to different sugars.These results indicate that YdGal-3,YdGal-9,and YdGal-1 played important roles in the innate immunity of N.albiflora.The antimicrobial peptide gene YdNkl-1 encoding 158 amino acids and the YdNkl-2encoding 151 amino acids.Both YdNkl-1 and YdNkl-2 have a signal peptide and a saposin B domain.The results of q RT-PCR showed that the m RNAs of YdNkl-1 and YdNkl-2 were widely expressed in 12 tissues tissues of N.albiflora.The highest expression level of YdNkl-1 was in skin,the higher expression levels of YdNkl-2 were in gill and spleen.After V.harveyi infection,the expression levels of YdNkl-1 in liver and spleen were significantly increased;and the expression levels of YdNkl-2 in head kidney,liver and spleen were all significantly increased.The subcellular localization results showed that YdNkl-1 and YdNkl-2 proteins were distributed in the cytoplasm and nucleus in HEK 293T cell.In addition,His-YdNkl-1 protein was purified by prokaryotic expression,laying a foundation for further research on the function of YdNkl-1and development of immunopotentiator.