Screening And Expression Analysis Of Genes Related To Vibrio Harveyi Resistance In Chinese Tongue Sole (Cynoglossus Semilaevis) Based On Transcriptomics

Vibrio harveyi is one of the main pathogens causing fatal vibriosis in Chinese tongue sole(Cynoglossus semilaevis),which seriously hampers the development of factory culture of C.semilaevis.In this study,we performed transcriptome analysis of C.semilaevis between resistant family and susceptible family under V.harveyi challenging and control conditions using high-throughput sequencing technology,aiming at screening candidate genes related to the immune response to V.harveyi,and obtaining a large amount of genes related to disease resistance.(1).We performed transcriptome sequencing of twelve C.semilaevis samples from four groups on the Illumina HiSeq platform.A total of 1.095 billion clean reads were obtained from 12 RNA-seq libraries,and 71.32% of the reads were successfully aligned to the reference genome using TopHat.A total of 23,088 genes were annotated with an average length of 2,485 bp.Additionally,we obtained 805 novel transcripts and 57,521 alternative splicing sites from 12 RNA-seq libraries.Furthermore,a total of 495,195 SNP and 45,480 InDel were obtained.We used HTSeq and R to calculate the FPKM of each gene and the distribution of FPKM from 12 RNA-seq libraries.(2).We compared gene expression levels under different experimental conditions,and the final FPKM of each group is the average of three biological replicates.We compared gene expression levels of four C.semilaevis groups and obtained 2,674 differentially expressed genes from four pairwise comparisons(CsRC vs CsRU,CsRC vs CsSC,CsRU vs CsSU,CsSC vs CsSU): the DEGs in CsRC and CsRU comparison comprising 684 up-regulated genes and 268 down-regulated genes,the DEGs in CsRC and CsSC comparison comprising 108 up-regulated genes and 396 down-regulated genes,the DEGs in CsRU and CsSU comparison comprising 635 upregulated genes and 408 down-regulated genes,the DEGs in CsSC and CsSU comparison comprising 25 up-regulated genes and 150 down-regulated genes.GO enrichment analysis showed that 6 DEGs(apc2,ccl19,ccl20,il-8,vnt,ercc-6)in CsRC and CsRU comparison,3 DEGs(apc2,dmbt1,etc.)in CsRC and CsSC comparison,9 DEGs(st3gal4,c1 q,tnfα1,etc.)in CsRU and CsSU comparison and 3 DEGs(ccl18,ccl20,c1qtnf9a)in CsSC and CsSU comparison,were annotated with immune response term.KEGG pathway enrichment analysis revealed that multiple DEGs were enriched in Jak-STAT signaling pathway,NOD-like receptor signaling pathway,RIG-I-like receptor signaling pathway,Toll-like receptor signaling pathway and Cytokine-cytokine receptor interaction.We identified 11 immune-related genes(tlr5,irak4,il-22rα2,lifr,pias1,etc.)in CsRC and CsRU comparison,2 immunerelated genes(il-6rα,pias2)in CsRC and CsSC comparison,25 immune-related genes(il-10,epor,gp130,prlr,jak2,stat5 b,etc.)in CsRU and CsSU comparison and 3 immune-related genes(socs7,ccl20,tgfβ2)in CsSC and CsSU comparison.Additionally,the FastQC,correlation test of biological replicates,and qPCR validation indicated that we presented a high quality transcriptomic dataset.(3).LncRNA plays an important role in the regulation of epigenetics,transcription,and post-transcription.To understand the regulation mechanism of immune response to Vibrio harveyi,we performed lncRNA identification and differential expression analysis from RNA-seq data.We obtained a total of 4,584 lncRNA loci,containing 5,714 transcripts in this study.The comparison of basic characteristics between lncRNA and the coding gene showed that the GC content of lncRNAs was lower than that of coding genes,the average exon length of lncRNAs was longer than that of coding genes,and the average expression of lncRNAs was slightly lower than coding genes.We screened 818,813,261 and 140 differentially expressed lncRNAs by differential expression analysis in four comparisons of Cynoglossus semilaevis groups(CsRU vs CsSU,CsRC vs CsSC,CsRC vs CsRU,CsSC vs CsSU),respectively.Additionally,we preliminarily analyzed the expression patterns and the relationship of differential expression of lncRNAs from those four groups.The qRT-PCR confirmed that the expressin patterns of lncRNAs were consistent with their expression levels based on transcriptome data.This study provides a large amount of reference data for studying the role of lncRNA in the immune response against V.harveyi.(4)We screened the IL-6 receptor from differential expression analysis,including IL-6Rα and GP130.To analyze the expression profiling of il-6rα and gp130 after Vibrio harveyi infection,we cloned and identified the full-length of il-6rα and gp130.We determined that the expressions of il-6rα and gp130 were relatively high in the liver,but low in the muscle.Time-course expression analysis revealed the upregulation of il-6rα and gp130 expressions in the kidney,spleen and liver after V.harveyi infection,which indicated that these genes were responsive to bacterial infection.It was worth noting that il-6rα and gp130 expression patterns were very similar.For example,both of them reached peak levels at 72 h in the kidney and at 48 h in the spleen.In the liver,il-6rα and gp130 remained stable over most of the time,but exhibited obvious upregulation at 24 h and 72 h,respectively.To understand the roles of IL-6Rα and GP130 in disease resistance,we analyzed the differential expression of il-6rα and gp130 between DS_CS and DR_CS in the liver,kidney,and spleen,and il-6rα and gp130 were differentially expressed only in the liver of DR_CS and DS_CS.