Molecular Cloning Of IgD And IgT,Analysis Of The IgH Locus And Immune Response In Largemouth Bass (Micropterus Salmoides)

Originated from the North America,Largemouth bass(Micropterus salmoides)is well-known by the short growth cycle and no inter muscle thorns.Largemouth bass has a wide range of habitats,as large as major reservoirs or lakes,and as small as ponds or streams.However,due to its complex living environment,the largemouth bass farming is affected by diseases.Among them,viruses and bacteria are the main pathogens of diseases.In order to improve aquaculture production,besides optimizing the living environment of the largemouth bass,intensive studies of the interaction between host and pathogens are crucial to further develop better vaccines.To understand the host immune response to pathogens,the full-length of IgD and IgT genes were first cloned,the main humoral immune effector molecules in largemouth bass.Furthermore,the organization of immunoglobulin heavy chain locus in largemouth bass was analyzed,which facilitates the study of antibody diversity.At last,the pathological changes of the largemouth bass mucosal tissues and the transcriptomic analysis in the gill and spleen were observed by using the GFP-Flavobacterium columnare immerse infection.The major contents and results are as follows:(1)According to the NCBI,a part of the IgD and IgT genes were found from the largemouth bass genome.The full-length IgD and IgT genes were obtained after RACE-PCR.The IgD gene was 3,243 bp with 3,000 bp ORF and predicted to encode999 amino acids with a transmembrane region.The IgT of the largemouth bass gene was 1,974 bp with 1,668 bp ORF(Open reading frame)and predicted to encode 555 amino acids containing a secreted tail.Furthermore,these two genes both have a signal peptide sequence that promotes the protein transmembrane and many cysteines that maintain the advanced structure of the protein.Then,sequence alignment showed that the largemouth bass IgD and IgT amino acid sequences were similar to most teleosts.There are seven constant regions in IgD with no exon replication existed,and four constant regions in IgT.The kinship of three immunoglobulins in bony fish was investigated the phylogenetic tree.The result revealed that IgM,IgD and IgT clustered into one branch with Perciformes.Finally,the expression of IgD and IgT genes in different tissues of largemouth bass were detected and found that these two genes showed higher expression in both spleen and head kidney.However,IgT expression in mucosal tissues was higher than IgD.(2)Immunoglobulins enable to recognition of many different antigens by V-D-J recombination,the structure of IgH locus help us further explor the recognition mechanism between antibodies and antigens.In this study,the segments encoding immunoglobulin were found in the largemouth bass genome by the obtained gene sequences.The Largemouth bass IgH locus was mapped based on the characteristics of the immunoglobulin variable regions,diversity regions,joining regions,and constant regions.The IgH locus of the largemouth bass was V26-Dτ3-Jτ5-Cτ-Dμ6-Jμ6-Cμ-Cδ.In addition,the 26 VH amino acid sequences were classified into three subgroups,and every single subgroup was over 80% sequence similarity.(3)To explore the immune response of largemouth bass after pathogens stimulation,largemouth bass were infected by immersion with GFP-Flavobacterium columnare.The result showed substantial bacterial were attached on the surface of the gill and skin,especially the gill after 1 day-post-infection(dpi).HE staining revealed that The thickening of primary lamellae was observed during the early phase of infection in largemouth bass,but gradually recovered after 14 dpi.The epidermis of skin was thickened after 4 dpi and then was significantly thinner than that in normal fish after 21 dpi.The phenomenon may be affected by the chondroitin lyase produced by bacteria may cause the exfoliation of epithelial cells by degrading acidic polysaccharides in the skin.In addition,q PCR showed that the expression of largemouth bass Igs differed in gill,skin,spleen and head and kidney at different periods of infection,indicating that all three immunoglobulins were involved in systemic and mucosal immunity to some extent.In conclusion,our study cloned the full-length cDNA sequences of IgD and IgT of largemouth bass for the first time,and the structure of the immunoglobulin heavy chain locus was analyzed,which is beneficial to explore the mechanism of antigen-antibody recognition.Finally,the expression of immunoglobulins was explored after bacterial infection.This study sheds new light on the interaction between the immunoglobulins and the mucosal immunity.