Molecular Cloning And Expression Analysis Of ?? TCR Genes In Dojo Loach(Misgurnus Anguillicaudatus)

Dojo loach?Misgurnus anguillicaudatus?is considered to be an important commercial species for aquaculture and distributes widely distributed in China,Japan and other Southeast Asian countries.Recently,the market is increasingly demanding loach,the price has risen,and the enthusiasm of farmed loach everywhere is also rising,but as the scale of loach,farming expands,high stocking density and deteriorating aquaculture environments have resulted in frequently infectious diseases and finally great economic losses.T cell receptor?TCR?plays a crucial role in immune system,which due to its important role in the recognition of pathogen invasion and in the activation of immune related pathways downstream.To date,little is known about the immunological function of??TCR genes after pathogenic challenges in loach.In this work,we firstly cloned and identified the full-length cDNA of TCR?and?chains from the loach spleen,and then their molecular characterization and expression profiles in early embryonic stages and different tissues were investigated from M.anguillicaudatus.Three infection models of dojo loach with F.columnare G₄,I.multifiliis and Saprolegnia sp.were constructed with obvious morphological changes of gills and skin.In addition,the expression levels of Ma-TCR?and Ma-TCR?were examined after challenge with bacterial,parasitic and fungus,constructed a heat map of gene expression.1.Based on partial cDNA fragments of TCR?and?genes were obtained from M.anguillicaudatus transcriptome database,the two chains of T cell receptor?TCR?in the loach were identified by PCR and cloned by RACE-PCR.The full-length cDNAs of Ma-TCR?and Ma-TCR?include an open reading frame?ORF?of 948 and 867 bp encoding a polypeptide of 316 and 288 amino acids,respectively.Structural analysis indicated that Ma-TCR?and Ma-TCR?had a signal peptide,a connecting peptide?CPS?,a transmembrane region?TM?and a cytoplasmic?CYT?and two Ig domains,which are similar to their counterparts described in other teleost.Multiple sequence alignment and phylogenetic analysis supported that dojo loach is closely related to the Cyprinidae family?Yu et al 2019?.The mRNA expression levels of the four TCR genes were detected in liver,spleen,kidney,skin,gill,intestine,eye,brain,blood,muscle,fin,fat and gonad.The highest relative expression of Ma-TCR?was detected in kidney,followed by spleen,gill,fin,brain.The highest relative expression of Ma-TCR?was detected in the gill while the lowest expression level was observed in muscle.2.In the present study,challenge with F.columnare G₄?1×10⁵ CFU/ml?,I.multifiliis?with the ratio of 10000 Ich theronts per fish?and Saprolegnia sp.?1×10⁵zoospore/ml?was performed that gill and skin of loach were observed obviously worsened after F.columnare G₄ and I.multifiliis infection.In infected loach,the length-width ratio of the secondary lamellae?SL?was detected smaller than control fish and a large number of goblet cells was greatly increased in skin.3.Three infection models of the loach with bacteria?F.columnare G₄?,parasite?Ichthyophthirius multifiliis?and fungus?Saprolegnia sp.?were constructed.After the infection changes in the expression patterns were detected in Ma-TCR?and Ma-TCR?by qRT-PCR.The morphological changes of gills and skin after challenged with F.columnare G₄ and Ichthyophthirius multifiliis were investigated.The results clearly indicated that Ma-TCR?and Ma-TCR?was significant up-regulated not only in spleen and kidney,but also in skin and gill.Compared to F.columnare G₄ infection,mRNA expression of both TCR?and TCR?showed higher sensitivity in classical immune organs?kidney and spleen?then mucosal tissues?skin and gill?after challenge with Ichthyophthirius multifiliis and Saprolegnia sp.Our results first indicated that TCR?and TCR?of dojo loach might function differently in response to challenge with different pathogens.In summary,our present findings suggested that Ma-TCR?and Ma-TCR?might play significantly roles in the modulation of immune response and protect loach from different pathogens infection.