| Flatfish belongs to Pleuronectiformes based on morphological classification,among which Chinese tongue sole(Cynoglossus semilaevis)is a benthic marine fish mainly distributed in coastal areas of China,such as the Bohai Sea and the Yellow Sea.C.semilaevis has very high economic value,as well as scientific value.In order to better adapt to benthic lifestyles,C.semilaevis has evolved a number of biological characteristics,such as smaller internal organs,less exercise,faster growth and less occupied water space.In addition,due to its delicious and smooth meat taste,high protein content,nutrient-rich,high market prices,good economic returns and other characteristics,it is favorited by fish farmers and thus become an important economic fish in China.However,during the breeding process of C.semilaevis,Vibrio harveyi is one major pathogen that leads to bacterial disease and affects the economic productivity,which in turn hinders the development of its aquaculture.In this study,we have conducted the locus mapping for resistance to V.harveyi,screened immune candidate genes and initiated functional analysis of these genes in Chinese tongue sole.It will provide a basis for further analysis of the resistance mechanisms to V.harveyi in Chinese tongue sole.1.Locus mapping for resistance to V.harveyi,screening of immune-related genesBased on the previous studies in our laboratory,the locus qE for resistance to V.harveyi located on chromosome 17(LG18 linkage group)has been narrowed from 7.6 Mb to 5.6 Mb,in order to facilitate the screening of disease-resistant and immune-related candidate genes.11 immune-related loci were located in integrate linkage group(LG 18),female linkage group(LG18F),and male linkage group(LG18M)in Chinese tongue sole.After transformation to physical region,311 genes were detected in this region.A total of 13 immune-related GO terms were enriched and 5 immune-related pathways were found by KEGG analysis.Finally,12 candidate genes were selected in combination with the functional classification of 311 genes,and two of them(dctn5 and stat5bl)were further studied in the subsequent experiments.These data provide the molecular resource for further study on the disease-resistant mechanism of Chinese tongue sole.2.Cloning,characterization analysis of dctn5,and preliminary functional analysis of dctn5_tv1 in immune response of Chinese tongue soleIn order to evaluate the function of dctn5 in the immune response of Chinese tongue sole,the full-length cDNAs of two dctn5 variants(named dctn5_tv1 and dctn5_tv2,respectively)were cloned and identified.The two variants were produced by alternatively splicing out the first(dctn5_tv1)and second(dctn5_tv2)exon.The qRT-PCR results showed that the dctn5_tv1 mRNA was widely expressed in immune-related tissues,while the dctn5_tv2 was mainly expressed in gonads.Based on these results,we proposed that dctn5_tv1 may play role in the immune response of Chinese tongue sole.Therefore,we further examined the expression levels of dctn5_tv1 in immune-related tissues after V.harveyi infection.The results revealed that dctn5_tv1 mRNA levels were up-regulated in gill,intestine,spleen,kidney and skin after V.harveyi challenge,supporting that dctn5_tv1 was involved in immune response of tongue sole.Furthermore,the recombinant Dctn5_tv1 protein was expressed,and its antimicrobial function in vitro was determined.The recombinant Dctn5_tv1 possessed the different antibacterial activity against Escherichia coli,Streptococcus agalactiae,Vibrio parahaemolyticus,Shewanella algae,and V.harveyi,while the high antibacterial activity were observed against E.coli and S.agalactiae.The bacterial binding assays showed that Dctn5_tv1 could bind to all tested bacteria cells,suggesting that the antimicrobial activity was probably synergetic with the binding to bacteria cells.These findings provided the study basis for exploring the disease-resistant mechanism of dctn5 in Chinese tongue sole.3.Cloning,characterization of stat5bl,and expressional pattern analysis in immune response of Chinese tongue soleIn order to evaluate the role of stat5bl in the immune response of Chinese tongue sole,the full-length cDNA of stat5bl was cloned.Multiple alignments of amino acid sequences showed that stat5bl and stat5b genes in teleosts shared a high similarity.In addition,the stat5bl and stat5b were clustered into the same branch in teleosts.Accordingly,we speculated that stat5bl and stat5b should be the same genes in teleosts.The qRT-PCR results showed that stat5bl mRNA was highly expressed in immune-related tissues,e.g.liver,gill,and skin,indicating the role of stat5bl in the immune response of Chinese tongue sole.To verify the role of stat5bl in the immune response,the expression of stat5bl in immune-related tissues was further examined after V harveyi infection.The results showed that stat5bl mRNA were up-regulated in spleen,gill,intestine,and skin after V harveyi challenge,supporting that stat5bl was involved in immune response of tongue sole.In addition,the knockdown of stat5bl gene in hepatocytes resulted in the regulation of several immune-related genes from the JAK-STAT signaling pathway.These results preliminarily uncovered the role of stat5bl gene in the immune response of Chinese tongue sole.4.Molecular characterization,expression,and preliminary functional analysis of a novel r-spondin member(rspo2l)in Chinese tongue soleIn addition to the functional studies of immune-related genes screened by QTL mapping,the functional studies of rspo2l genes in the R-spondins family screened by our laboratory through transcriptome analysis.A new member of the r-spondin family(rspo2l)was identified in Chinese tongue sole.In order to study the function of rspo2l,we cloned the full-length cDNA of rspo2l.Phylogenetic analysis showed that the rspo2l genes clustered into one group,while rspo2 was found to cluster into another group,suggesting that rspo2l has a different evolutionary status compared to rspo2,and further illustrating that rspo2l and rspo2 are two independent genes.The expression pattern of rspo2l showed that rspo2l mRNA was highly expressed in gill,skin,and liver,and low expressed in other tissues.In addition,the expression levels of rspo2l and its downstream gene,?-catenin,were examined in immune-related tissues after V.harveyi infection.The results showed that rspo2l mRNA levels were up-regulated in gill,skin,spleen,and kidney,supporting for the involvement of rspo2l in immune response of tongue sole.Moreover,?-catenin exhibited similar expression pattern to rspo2l after V.harveyi,suggesting a possible correlation between immune response of rspo2l and Wnt/?-catenin signaling pathway.Furthermore,the recombinant Rspo21 protein was expressed,and its antimicrobial function in vitro was also analyzed.The recombinant Rspo21 displayed no significant antibacterial activity against E.coli,V.parahaemolyticus,and V.harveyi.Based on these data,we speculated that Rspo21 was not directly but indirectly involved in immune response of tongue sole by the regulating Wnt/?-catenin signaling pathway.These data preliminarily explained the role of rspo2l in the immune response of Chinese tongue sole. |
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