F-type lectins(Fucolectins)are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion.In our previous research,we found that two homologous Fucolectin g...F-type lectins(Fucolectins)are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion.In our previous research,we found that two homologous Fucolectin genes,AjFTL-1 and AjFTL-2,exhibited different expression profiles after lipopolysaccharides(LPS)challenge in Apostichopus japonicus.However,the transcriptional regulation mechanism of these two genes remains largely unknown.In this study,the 5'flanking regions of Aj FTL-1 and Aj FTL-2 genes were cloned and the promoter activities were studied in epithelioma papulosum cyprinid(EPC)cell system.First,in silico analysis indicated that these two promoters both contain numerous putative transcription factor binding sites including NF-κB,CREB,and CREBP1,and both contain a TATA box.Additionally,luciferase assay and progressive 5'truncation analysis revealed that AjFTL-1 and AjFTL-2 both possess high promoter activities in EPC cells.Moreover,the luciferase activity of AjFTL-1 promoter was significantly regulated by peptidoglycan(PGN)and mannan(MAN),while AjFTL-2 promoter was prominently regulated by LPS and MAN,indicating Aj FTL-1 and Aj FTL-2 genes showed different transcriptional regulation pattern under different immune stimulation.More importantly,analyses of the functional promoter regions revealed the presence of two potential NF-κB binding sites(-769 bp to-761 bp,-185 bp to-172 bp)in Aj FTL-1 and one potential binding site(-530 to-517 bp)in Aj FTL-2.Different truncated reporter vectors and expression vector co-transfection revealed that transcription factor NF-κB/Rel could significantly increase the AjFTL-2 promoter activity,but not AjFTL-1 promoter activity.These findings indicated that in marine invertebrates,different Fucolectin members differ in transcription regulations and expression patterns,and might play different roles in immune defenses during pathogen infection.展开更多
基金financially supported by the National Natural Science Foundation of China(No.31802331)the Natural Science Foundation of Zhejiang Province(Nos.LZ19C190001,LY20C190002)+1 种基金the Natural Science Foundation of Ningbo(No.2018A610340)the K.C.Wong Magna Fund in Ningbo University。
文摘F-type lectins(Fucolectins)are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion.In our previous research,we found that two homologous Fucolectin genes,AjFTL-1 and AjFTL-2,exhibited different expression profiles after lipopolysaccharides(LPS)challenge in Apostichopus japonicus.However,the transcriptional regulation mechanism of these two genes remains largely unknown.In this study,the 5'flanking regions of Aj FTL-1 and Aj FTL-2 genes were cloned and the promoter activities were studied in epithelioma papulosum cyprinid(EPC)cell system.First,in silico analysis indicated that these two promoters both contain numerous putative transcription factor binding sites including NF-κB,CREB,and CREBP1,and both contain a TATA box.Additionally,luciferase assay and progressive 5'truncation analysis revealed that AjFTL-1 and AjFTL-2 both possess high promoter activities in EPC cells.Moreover,the luciferase activity of AjFTL-1 promoter was significantly regulated by peptidoglycan(PGN)and mannan(MAN),while AjFTL-2 promoter was prominently regulated by LPS and MAN,indicating Aj FTL-1 and Aj FTL-2 genes showed different transcriptional regulation pattern under different immune stimulation.More importantly,analyses of the functional promoter regions revealed the presence of two potential NF-κB binding sites(-769 bp to-761 bp,-185 bp to-172 bp)in Aj FTL-1 and one potential binding site(-530 to-517 bp)in Aj FTL-2.Different truncated reporter vectors and expression vector co-transfection revealed that transcription factor NF-κB/Rel could significantly increase the AjFTL-2 promoter activity,but not AjFTL-1 promoter activity.These findings indicated that in marine invertebrates,different Fucolectin members differ in transcription regulations and expression patterns,and might play different roles in immune defenses during pathogen infection.
