| Japanese flounder is one of the important types of mariculture in China In recent years,frequent severe viral diseases such as Lymphocystis disease were breeding industry sustainable development,make the farmers suffered a serious economic loss,cause the Japanese flounder breeding area,yield by the development of biotechnology,impetus the development of modern seed industry,has become a new methods of breeding.Splicing is move introns except show the son together is assembled into mature m RNA,the process of alternative splicing is found with splicing,more and more studies have found that alternative splicing is widespread in multicellular eukaryotes,it allows a gene produce multiple m RNA subtypes,and encoding proteins with different functions,increase the diversity of the proteome,but normal breaks in splicing patterns can lead to genetic disorders or diseases Triple motif(TRIM)and TRIM-like protein is a highly common protein family,their features is a conservative RING-BBox-coiled coil(RBCC)motif,there are one or more of the c-terminal domain structure unique TRIM proteins regulate various cellular processes,including transcription,signal transduction,cell fate and cycle also decided to TRIM family because of a RING structure domain and E3 ubiquitin ligase activity Many human TRIM protein expression can be induced by interferon or virus infection,can play a antiviral protein function TRIM at least three main antiviral mechanism have been reported.In this study,two genes closely related to disease resistance,thbs2 and trim25,were screened out,and molecular identification of thbs2 and trim25 genes in Japanese flounder was conducted by using molecular biology methods,and the mechanism of disease resistance of Trim25 was studied.The specific research contents are as follows:1.Identification and expression analysis of thbs2 and trim25 in Japanese flounderHomologous sequences of thbs2 and trim25 were identified from transcriptome data of the head kidney of Japanese flounder,and their full length was amplified by RACE method.The research found that there were four alternative spliced variants in Japanese flounder trim25,which were trim25 X1、trim25 X2、trim25 X3、trim25 X4.The nucleotide and amino acid sequences were analyzed to predict the protein domain and construct phylogenetic tree using bioinformatics website and software.Fluorescence quantitative PCR was used to analyze the m RNA relative expression levels at different embryonic development stages.The c DNA length of thbs2 gene was 4313 bp,including 210 bp 5 ’UTR length and 575 bp 3’ UTR length,with 10 conserved domains.The gene was most closely related to Larimichthys crocea and Lates calcarifer,and its expression level was significantly increased during the heartbeat and membrane exudation stages.The amino acid sequence homology of the four alternative spliced variants of trim25 reached 95.49%.All have RING,BBOX,PRY/ SPY domains.trim25 X1 and trim25 X2 form a separate branch,which then clings together with trim25 X3 and trim25 X4.They are most similar to Lates calcarifer and Oreochromis niloticus.The expression levels of the four alternative spliced variants were higher at the early stage of embryonic development than at the late stage.2.The subcellular localization of thbs2 and trim25 and the correlation analysis of anti-lymphocyst in Japanese flounderTo further understand the correlation between thbs2 and trim25 and lymphocyst disease and the localization of cells in Japanese flounder.By seamless cloning,p EGFP-thbs2 and four eukaryotic expression vectors p EGFP-trim25 X1/X2/X3/X4 were constructed.The eukaryotic expression of the genes was observed by cell transfection and subcellular localization.Fluorescence quantitative PCR was used to detect the relative expression of genes in lymphocyst resistant and sensitive tissues of Japanese flounder.Finally,four eukaryotic expression vectors of thbs2 and trim25 four alternative spliced variants,were successfully constructed.Thbs2 was distributed throughout the cytoplasm.TRIM25 X1/X2/X3 was distributed punctatively in the cytoplasm and X5 was localized throughout the cytoplasm and nucleus.Fluorescent quantitative PCR showed that the expression of thbs2 in head kidney,liver,blood,gill and heart was significantly higher in resistant individuals than in diseased individuals,and the difference was extremely significant in muscle.Trim25 X1 was expressed more in disease-resistant tissues,and X2/X3/X4 was expressed more in sensitive gills,muscles,and hearts.These data indicated that the expression of thbs2 and trim25 differed in the disease resistant and sensitive tissues of Japanese flounder,suggesting that thbs2 and trim25 were correlated with the resistance to lymphocyst disease.3.Study on the function of TRIM25 in innate immune response of flounderIn order to further understand the role of the four alternative spliced variants in innate immune response,whether there are functional differences and the mechanism of action,we stimulated cells and in vivo with the virus simulant poly I:C,and collected samples to detect the expression levels of m RNA and protein.The relative expression levels of related genes were detected by fluorescence quantitative PCR in the collected samples.The results showed that the expression of the four alternative spliced variants was upregulated in the cells stimulated by poly I:C,but the expression of trim25 X1/X3 did not increase or decrease at five time points,while X2 and X4 showed an increasing trend at 60 h.WB results showed that the expression level of Trim25 X2 was low after 12 h of poly I:C stimulation,but significantly increased at24-60 h.Immune related genes showed different expression patterns in lymphocyst resistant and sensitive tissues of Japanese flacebot,and could be up-regulated to varying degrees by poly I:C stimulation.All of these genes are located in the RIG-I signaling pathway,and it is possible that TRIM25 connects with these immune-related genes through this signaling pathway and induces them to produce antiviral immune response together. |
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