Screening Of Genes Related To Resistance To Cryptocaryon Irritans Infection In Trachinotus Ovatus Based On Transcriptome Analysis

Trachinotus ovatus has become one of the most important seawater aquaculture fish species in the coastal areas of South China.The stimulation of Cryptocaryon irritans has always been a difficult problem for the development of T.ovatus aquaculture,and severely infected fish can experience massive deaths in a short period.However,there has been no safe and effective methods to deal with C.irritans infection.In this study,whole transcriptome analysis of the skin tissue of T.ovatus after infection with C.irritans was performed based on RNA sequencing(RNA-Seq)to analyze theimpact of C.irritans on T.ovatus at the transcription level.Significantly differentially expressed genes(DEGs)were selected,and structure and expression profiles of these genes were analyzed.At the same time,resistant and susceptible groups of T.ovatus against C.irritans were established,and SNP sites with siginificant association with C.irritans resistance were identified.Finally,the regulation relationships between miRNA and target genes were confirmed through the dualluciferase system.These results will provide important technical support for the genetic improvement of T.ovatus resistance to C.irritans.The main research results are as follows:(1)Transcriptome sequencing was performed on the skin tissues of healthy and C.irritans-infected T.ovatus.The results found that after infection with C.irritans,genes related to lysosomes,phagosomes and proteasomes etc changed significantly,indicating that innate immunity played an important role in the resistance of T.ovatus to parasite infection.The significantly changed miRNA and m RNA expression were analyzed by miRNA-m RNA joint analysis.GO enrichment analysis results showed that most DEGs were enriched in the biological process category.KEGG enrichment analysis mainly focused on pathways such as myocardial contraction,metabolic pathways,and adrenergic signaling in myocardial cells.(2)Matrix metalloproteinase 9(MMP9)and laminin gamma 2(LAMC2)were selected from the miRNA-m RNA joint analysis data.The ORF lengths of MMP9 and LAMC2 genes were 2058 bp and 3390 bp,respectively,encoding 685 and 1130 amino acids.Amino acid sequence structure prediction results showed that MMP9 contained three fibronectin 2-type structures(FN2)and four coagulation factor-like repeat structures(HX),while LAMC2 contained one spectrin repeat protein(SPEC)domain and four epidermal growth factor(EGF)domains.Gene structure analysis showed that the structures of MMP9 and LAMC2 proteins were relatively conserved among chordates.Phylogenetic tree results showed that fish genes were clustered in one main branch,with the closest evolutionary relationship to MMP9 being Seriola dumerili and Thunnus albacares,and the closest evolutionary relationship to LAMC2 being Seriola lalandi.(3)The tissue expression profile analysis results showed that MMP9 was differentially expressed in all detected tissues,with relatively high expression levels in the fins and gills.The q PCR results showed that both MMP9 and LAMC2 m RNA levels in the skin and gills of T.ovatus before and after infection with C.irritans had an upward trend followed by a downward trend,reaching the highest levels at 24 h.(4)A total of 2 SNP sites were screened in the MMP9 gene using the direct sequencing method,with SNP(-1340A/C)located in the promoter and SNP(+400A/G)located in the first intron.Gene genotyping and association analysis of SNPs with C.irritans resistance revealed a significant association with SNP(+400A/G).A total of 2SNP sites were screened in the LAMC2 gene,with SNP(-1785A/T)located in the promoter and SNP(+10336A/C)located in the twelfth intron,and SNP(-1785A/T)was significantly associated with C.irritans resistance.(5)Transcriptome data and software prediction results showed that miR-221 and miR-30 c might interact with MMP9,and miR-200 b and miR-27 d might interact with LAMC2.The dual-luciferase enzyme detection results further showed negative regulation relationship between miR-221 and MMP9,and miR-200 band LAMC2.