Transcriptomics And Proteomics Analyses Of High-resistance And Susceptible Families Of Genetically Improved Farmed Tilapia After Streptococcus Agalactiae Infection

Genetically improved farmed tilapia?GIFT?is Nile tilapia with genetically improved traits.GIFT has become the dominant species of tilapia in China because of its advantages of fast growth rate,high meat yield,and ease of catching.However,as a result of changes in cultivation mode and the environment,Streptococcus agalactiae infection has become a bottleneck restricting the sustainable development of the tilapia industry.Therefore,there is an urgent need to identify the immune response mechanism of GIFT following S.agalactiae infection,and to determine the regulatory network of key immune-activated genes with the aim of breeding new resistant strains through molecular marker-assisted selection or gene editing.In the present study,GIFT families were established by pair-mating.High-resistance and susceptible families were evaluated by artificial injection of S.agalactiae and the detection of immune-related enzyme activity in the liver.RNA sequencing?RNA-seq?and isobaric tags for relative and absolute quantitation?iTRAQ?technology were used to sequence transcripts and determine proteomics of the spleen in high-resistance and susceptible families.The differential expression of genes associated with the immune response was screened out by the combined analysis of transcriptome and proteomics,and candidate genes for disease resistance were screened and validated.The main findings were as follows:1.Family establishment and disease resistance of different families of GIFTFifty-three families of GIFT were established by pair-mating,and each family was challenged by S.agalactiae?HN016 strain?.Survival data showed that 12 families had strong disease resistance with a survival rate over 90%;14 families had strong disease resistance with a survival rate of 70%-89%;19 families had common disease resistance with a survival rate of 30%-69%;and eight families had low disease resistance with a survival rate of less than 30%.The activities of superoxide dismutase,alkaline phosphatase,lysozyme,and total anti-oxidant capacity in the liver of high-resistance and susceptible families were compared after the S.agalactiae challenge.The activities of these four enzymes were up-regulated in high resistance families during the early stage of infection,but down-regulated in susceptible families.This indicated that the activities of these four enzymes are associated with disease resistance in GIFT.2.Transcriptome analysis of the spleen in GIFT families with different disease resistance after S.agalactiae infectionRNA-seq was used to analyze differences in gene expression in the spleen of GIFT families with different levels of disease resistance at four stages?0h,5 h,50 h,and 7d?after S.agalactiae infection.A total of 5756 differentially expressed genes were identified,including 1022 in the 0 h group,4937 in the 5 h group,267 in the 50 h group,and 259 in the 7 d group.Functional annotations of these genes revealed a strong ability in pathogen identification,antigen presentation,and immune activation in GIFT families with high disease resistance,while susceptible families showed a strong ability in apoptosis activation.Among the 5756 differentially expressed genes,we screened out seven?encoding cathepsin,chemokine,integrin,interleukin,lysozyme C,major histocompatibility complex?MHC?,and galectin?potential candidate genes of disease resistance in GIFT.3.Proteome analysis of the spleen in GIFT families with different disease resistance after S.agalactiae infectioniTRAQ was applied to analyze differences in protein expression in the spleen of high disease resistance and susceptible GIFT families 0 h,5 h,50 h,and 7 d after S.agalactiae infection.A total of 1607 protein types were identified at the four different stages.Of these,105 were up-regulated and 68 were down-regulated at 0 h post infection,and 108 were up-regulated and 75 were down-regulated at 5 h post infection.Combined analysis of transcriptomes and proteomics revealed that the expression pattern of 24 and 41 genes were changed at both the mRNA level and protein level at 5 h and 50h post-infection,relatively.These differentially expressed proteins participate in many pathways such as energy metabolism,the immune response,cytoskeleton formation,vesicle transport,post-translational modification,and nucleic acid transport and metabolism.Among these proteins,cathepsin B,galectin-3,MHC class I,CC chemokine,and complement C1r may be candidate genes for anti-Streptococcus in GIFT.4.Cloning and immune response analysis of cathepsin B as a candidate geneThe full-length cDNA of the cathepsin B gene was obtained by rapid amplification of cDNA ends?RACE?and PCR.The full-length cathepsin B cDNA is 1746 bp,its 5'untranslated region?UTR?is 161 bp,and its 3'UTR is 592 bp.The open reading frame?ORF?is 993 bp,which encodes a total of 331 amino acids.Cathepsin B gene expression in healthy fish was highest in the gills,following by the intestines and spleen.After infection with S.aga.lactiae,cathepsin B expression was clearly up-regulated in the spleen,head,kidney,and intestines 5 h and 50 h post-infection in high-resistance and susceptible families.Cathepsin B expression was up-regulated to a significantly higher extent in the high-resistance GIFT family than the susceptible family at the same time and in the same tissues?P<0.05?.These results suggest that cathepsin B plays an important role in the immune response to S.agalactiae infection in GIFT.5.Cloning and immune response analysis of the galectin-3 gene in GIFTThe full-length cDNA of the galectin-3 gene was obtained by RACE and PCR.The full-length galectin-3 cDNA is 1034 bp,its 5'UTR is 182 bp,and its 3' UTR is 162 bp.It has a 690 bp ORF,including five exons and four introns,encoding 229 amino acids with a predicted molecular weight of 24.81 kDa and an isoelectric point of 7.98.The galectin-3 sequence was uploaded to the GenBank database under accession number NC₀31983.1.Galectin-3 gene expression in healthy fish was highest in the spleen and kidney.After S.agalactiae infection,expression was significantly up-regulated in immune-related tissues?such as the intestine,liver,spleen,anterior kidney,and gills?.Peak expression in the spleen,anterior kidney,and gills was 5 h post infection,that in the intestine was 50 h,and that in the liver was 7 d.Galectin-3 gene expression was also clearly up-regulated in the intestine,liver,spleen,kidney,and gills after Aeromonas hydrophila infection,with peak expression 18 h post-infection in the intestine,liver,and gills,12 h in the kidney,and 36 h in the spleen.These results suggest that galectin-3 plays an important role in the immune response of GIFT after bacterial infectionIn conclusion,we screened high resistance and susceptible families of GIFT,and compared the expression of transcripts and proteomics in the spleen after S.agalactiae challenge.The immune response mechanism of GIFT to S,agalactiae infection was revealed at the level of both the gene and the protein Cathepsin B and galectin 3 were preliminarily identified as anti-Streptococcus agalactiae genes in GIFT.These findings aid future work in molecular marker-assisted breeding selection and vaccine research and development.