Screening And Identification Of Monopterus Albus Hemorrhagic Disorder Resistance Genes

Monopterus albus is a freshwater farmed fish with broad market prospects.Due to its tender meat and rich nutrition,the scale of its aquaculture continues to expand.In recent years,with the development of breeding technology and the expansion of breeding scale,the outbreak of Monopterus albus disease has become one of the "bottlenecks" in the development of breeding.Hemorrhagic disease is a common disease in the cultivation of Monopterus albus and it has been identified that the pathogen of this disease is mainly Aeromonas hydrophila.It is extremely necessary to explore the response mechanism of immune-related genes of Monopterus albus after infection with Aeromonas hydrophila.We took the liver,spleen,intestine and skin of the Monopterus albus after 48 hours of infection with Aeromonas hydrophila,and completed the transcriptome sequencing through the Illumina sequencing platform.In this sequencing,a total of40376 differential genes were detected,of which 9379,13492,7645 and 9860 differential genes were detected in the intestine,skin,liver and spleen respectively.We divided the four tissues into systemic immunity group(including liver and spleen)and mucosal immunity group(including intestine and skin)and performed KEGG enrichment analysis on their differential genes.The systemic immune group shared2124 differential genes which were enriched in 17 immune-related pathways;the mucosal immune group shared 2813 differential genes which were enriched in a total of 18 immune-related pathways.Based on the above transcriptome analysis results.WGCNA(weighted gene co-expression network analysis)was used to construct a gene co-expression network.Enter the expression matrix of all differential genes,calculate and determine the soft threshold β=20 and calculate the differential genes this time and cluster them into 20 modules.Among them,magenta,floralwhite and darkred which are highly correlated with Aeromonas hydrophila infection were identified.The Perl language was used to calculate the core genes in these 3 modules,and the top 15% of the genes in connectivity were used as key genes.We take the intersection of statistical key genes and related immune genes enriched by KEGG as candidate genes.Candidate genes for systemic immune organs include:IL-6R,C3,CCL-3,MHC-I and TNF-α;Candidate genes for mucosal immune organs include:CD-22,IL-6R,MR.We cloned the MR of Monopterus albus and named it MaMR.Through SMART domain prediction analysis,it is found that the extracellular region of MaMR contains1 CR(Cysteine-rich domain)domain,1 FN(Fibronectin domain)domain and 8tandem CTLD(C-type lectin-like domain).We respectively carried out prokaryotic expression and protein purification of different domains of CR,FN and CTLD4-8 of MaMR and analyzed the agglutination effect of different domains on bacteria.It was found that the CR domain can agglutinate Klebsiella pneumoniae and Aeromonas hydrophila,CTLD4-8 can agglutinate Klebsiella pneumoniae,Bacillus megaterium,Pseudomonas aeruginosa and Aeromonas hydrophila.The FN domain can not agglutinate the above bacteria.Subsequently,we tested the sugar binding ability of different domains by ELISA.The results show that CTLD4-8 can bind to glucose,lipopolysaccharide,mannose and rhamnose while the CR and FN domains can not bind to the above four sugars.The intracellular domain of MaMR has no signal transduction function and requires the assistance of other receptors.Through STRING database analysis,it has found that MR and TLR2 interact.In order to verify the interaction between the two genes,we constructed the expression vectors of MaMR and Ma TLR2 and co-transfected them into EPC cells.Immunofluorescence results showed that MaMR and Ma TLR2 can co-localize.The luciferase reporter gene experiment was used to detect the effects of MaMR and Ma TLR2 on transcription factor activation.The results showed that in the cells co-transfected with MaMR and Ma TLR2,the activity of AP-1 was significantly higher than that of MaMR or Ma TLR2 transfected alone.In EPC cells,the expression of IL-1β,IL-8 and TNF-αincreased significantly after MaMR and Ma TLR2 were overexpressed at the same time.WGCNA analysis can make the network relationship of genes conform to the non-scale distribution by power processing the correlation coefficient and more truly restore the biological process of the Monopterus albus infected with Aeromonas hydrophila.This study is based on transcriptome analysis,using WGCNA to construct a gene co-expression network and systematically analyze the immune response mechanism of Monopterus albus after infection with Aeromonas hydrophila.The results of this study can lay a theoretical basis for the follow-up study of the anti-bacterial immune response mechanism of Monopterus albus.