Identification And Immune Function Study Of Cynoglossus Semilaevis Perforin-1 Like Gene And Liver Transcriptome Analysis After Vibrio Harveyi Infection

Cynoglossus semilaevis is mainly distributed in the coastal areas of China and is one of the most important marine fishes in China.Because of its delicious taste and rich nutritional value,it is deeply loved by consumers and has good market value.In recent years,with the continuous expansion of aquaculture scale and the pollution of aquaculture water,diseases occur frequently and become more and more serious.Among them,skin ulcerative disease caused by Vibrio harveyi has created great economic losses to the C.semilaevis farming industry.For decades,the prevention and control of fish diseases has mainly relied on antibiotics.However,the traditional drug treatment can not only fail to solve the problem fundamentally,but also bring a series of new problems.Although a lot of research have done on the C.semilaevis disease resistance breeding,the basic research on disease resistance of C.semilaevis is very weak,which severely restricts the rapid development of C.semilaevis disease resistance breeding.In recent years,a batch of homologous genes of other animal immune-related genes in the C.semilaevis have been reported by homologous cloning technology,but these genes are not specific to the C.semilaevis disease treatment,and this method is also time-consuming and labor-intensive.Therefore,it is eager to use other methods to screen out disease-resistant genes that focus on the semi-smooth tongue sole itself,and then study the mechanism of disease resistance and immunity,so as to further explore their application in disease resistance breeding of C.semilaevis.In this paper,an important gene against V.harveyi was identified by genome-wide association analysis(GWAS)and its antimicrobial function was studied,the expression pattern of the synergistic gene of its was also analyzed.In addition,the C.semilaevis liver transcriptome of V.harveyi infection was also analyzed in this paper.The research content is as follows:1.Identification and immune functional analysis of perforin-1 like gene of C.semilaevisBased on the re-sequencing data of V.harveyi disease-resistant and disease-susceptible families established using old methods for many years,GWAS analysis was carried out in this paper.One of the significantly associated SNPs was identified within the C.semilaevis perforin-1 like gene(CsPRF1l)on chromosome 9(-log 10(_P-value)=6.29),and then the CsPRF1l sequence was verified and characterized.The full-length c DNA sequence of CsPRF1l was 1835 bp,and the open reading frame(ORF)of its was 1545 bp,encoding514 amino acids.CsPRF1l protein contains the conserved MACPF and C2 domains.Real-time qualitative polymerase chain reaction(q RT-PCR)showed that CsPRF1l presented a higher intestinal expression level in disease-resistant families than in susceptible families,CsPRF1l is widely distributed in most of the tested tissues of healthy C.semilaevis adults and highly expressed in the intestine,brain,stomach,and gills,and CsPRF1l m RNA was markedly upregulated in the liver,spleen,kidney,intestine,gills and skin at six time points after V.harveyi infection.Through the Oxford Cup inhibition zone experiment,it was found that the recombinant CsPRF1l protein in E.coli can significantly inhibit the growth of pathogenic bacteria in vitro.After injection with V.harveyi and r CsPRF1l protein,it was found that the r CsPRF1l protein had a significant immune protective effect on the zebrafish.These results provide theoretical basis and gene markers or gene products for disease-resistant breeding and disease prevention and control of C.semilaevis,which have important significance and application value for disease-resistant breeding of C.semilaevis.2.Construction of CsPRF1l transgenic zebrafish model and zebrafish perforin knockout modelIn this study,CsPRF1l transgenic expression vector was constructed and microinjected into zebrafish 1-cell stage embryos.Through fluorescence microscope observation,it was found that the CMV promoter can drive the strong expression of green fluorescent protein(GFP)throughout the body,and GFP continued to be expressed in embryos science the microinjection.GFP was also strongly expressed in F1 and F2CsPRF1l transgenic zebrafish embryos,and PCR identification confirmed that almost all the embryos expressing green fluorescent protein did integrate the CsPRF1l gene.The embryos expressing green fluorescent protein were selected to grow to adult fish,and their tail fin genomic DNA was identified by PCR,and the positive CsPRF1l transgenic zebrafish adults were selected for V.harveyi infection experiment.It was found that compared with the wild type zebrafish,the mortality rate of the CsPRF1l transgenic zebrafish was significantly lower,which indicated that the disease resistance of the CsPRF1l transgenic zebrafish was indeed improved.In addition,this paper also used CRISPR/Cas9 technology to knock out the zebrafish perforin gene(Dr PRF1.5).It was found that the germline heritability of P0 Dr PRF1.5 mutants was 56%,and 36.8%of the F1 Dr PRF1.5 mutants could produce effective mutations.The Dr PRF1.5 knockout model established in this study lays the foundation for subsequent screening of homozygotes and analyzing the immune function of Dr PRF1.5 gene.Therefore,based on the zebrafish gene editing model,we can not only quickly verify the immune function of CsPRF1l gene in fish,but also provide a reference for transgenic disease-resistant breeding in C.semilaevis.3.Characterization and expression analysis of C.semilaevis granzyme B geneC.semilaevis granzyme B gene(CsGzmBl)was first verified and analyzed in this paper.The full-length c DNA sequence of CsGzmBl was 923 bp,contained a 49 bp 5’UTR,a 94bp 3’UTR,and a 780 bp ORF region encoding 259 amino acids protein with 19 amino acid residues signal peptide.The genome structure of CsGzmBl is highly conserved,consisting of 5 exons and 4 introns.The CsGzmBl protein has two N-terminal glycosylation sites,a catalytic triad“His63Asp112Ser207”,a conserved“PHSRPYMA”amino acid sequence,six conservative cysteine(Cys)and other functional domains or sites.q RT-PCR indicated that CsGzmBl m RNA was highest expressed in spleen of healthy C.semilaevis adults,followed by head kidney,kidney,liver,and gills,and lowest in the muscle,and CsGzmBl m RNA in six tissues(spleen,intestine,liver,skin,gills and kidneys)was up-regulated to varying degrees after stimulation with V.harveyi.These results indicate that CsGzmBl gene can be induced by pathogens,and provide basic information for the further study of the antibacterial and immune function of the CsGzmBl gene.4.Transcriptome analysis of the C.semilaevis liver after V.harveyi infectionIn this study,the C.semilaevis liver transcriptome was sequenced and analyzed at 0 h,24 h,48 h and 96 h after V.harveyi infection.The high-quality reads obtained at each processing time point averaged 56462502,54556955,53307869,49203667,and the sequences aligned to exons reached more than 70%.Difference analysis showed that a total of 5155 differentially expressed genes were screened compared with 0 h.Most of these genes were involved in the synthesis of endoplasmic reticulum,protein N-terminal and O-terminal glycosylation modification,DNA replication,recombination and repair,and extracellular communication and other biological processes.Fifteen differential genes were selected for q RT-PCR,and it was shown that the accuracy of the sequencing results.Compared with 0 h,the m RNA expression levels of Ckm,Tgm2b,Hmx3a and Dnajb12b were significantly different at 24 h,48 h and 96 h after infection.This study provides more materials for further understanding of the relevant regulatory molecules of the immune response of C.semilaevis after infection with V.harveyi,and is conducive to the subsequent research on C.semilaevis disease-resistant breeding.