Characterization And Function Analysis Of Ube2g1 And C1qbp Genes In Large Yellow Croaker (Larimichthys Crocea)

Large yellow croaker(Larimichthys crocea),a yellow-golden colored fish,is a popular economically important marine species in China due to its high nutritional value and tender meat.Outbreaks of visceral white spot disease caused by Pseudomonas plecoglossicida can result in severe economic losses in its aquaculture.Based on the results of a previous genome-wide association study(GWAS),ubiquitin-conjugating enzyme E2G1(LcUbe2g1)and complement component 1q binding protein(LcC1qbp)were identified as two important candidate genes associated with the poor disease resistance in L.crocea.In this study,we investigated the molecular characteristics and functions of these two genes to explore their roles and mechanisms in the immune response of L.crocea against P.plecoglossicida.The main results are as follows:The open reading frame of LcUbe2g1 is 513 bp,encoding 170 amino acids.Real-time Quantitative PCR(RT-q PCR)analysis revealed that LcUbe2g1 is widely expressed in L.crocea,with the highest expression in brain.Upon immune stimulation with P.plecoglossicida,both the transcription and protein levels of LcUbe2g1 were significantly up-regulated,as demonstrated by RT-q PCR and immunohistochemistry.Subcellular localization analysis showed that LcUBE2G1 protein is distributed in both the cytoplasm and nucleus of L.crocea head-kidney cells,and immunocytochemical analysis indicated that LcUBE2G1 protein and ubiquitin(Ub)protein partially co-localize in these cells.Further analysis using GST pull-down,mass spectrometry,mammalian cell two-hybrid and point mutation assays revealed that LcUBE2G1 directly interacts with NEDD8,after modification,connects with Ub protein to exert its ubiquitin ligase function.The open reading frame of LcC1 qbp is 852 bp,encoding 283 amino acids.RT-q PCR analysis showed that LcC1 qbp is widely expressed in L.crocea,with the highest expression in kidney.Subcellular localization analysis revealed that LcC1 QBP protein is distributed in both the cytoplasm and nucleus of L.crocea head-kidney cells.Upon immune stimulation with P.plecoglossicida,both the transcription and protein levels of LcC1 qbp were significantly upregulated,as demonstrated by RT-q PCR and immunohistochemistry.Using RNA interference and transcriptome analysis in the HEK 293 T cell line,1327 differentially expressed genes were identified,including 821 up-regulated and 506 down-regulated genes.Among these,85 important immune-related genes directly associated with the complement system,including C1 s and C3 complement components,CXCR4/6,RIPK4/1,TLR6,and MYD88 were identified.GO and KEGG enrichment analysis revealed that these differentially expressed genes were mainly enriched in immune system development,MAPK signaling pathway,TNF signaling pathway,and C-type lectin receptor signaling pathway.This study aims to reveal the roles and mechanisms of two candidate genes,ubiquitinconjugating enzyme E2G1(LcUbe2g1)and complement component 1q binding protein(LcC1qbp),in the antibacterial immune response of large yellow croaker,as well as to provide new insights into the genetic basis and immune response mechanism of large yellow croaker against visceral white spot disease,and to offer references for disease-resistant molecular breeding.