Identification And Functional Study Of Three Variable Splices Of The Fthl27 Gene In Miichthys Miiuy

With the development of biotechnology and computer technology,the popularity of second-generation sequencing and the rise of third-generation sequencing technology,the research on variable splicing is also in full swing.Variable splicing produces highly different gene splicing fragments through different splicing methods,which is an important reason for the mechanism to regulate gene expression and lead to biological function differentiation,as well as to promote the generation of protein subtypes with different functions,This is the main reason for the expansion of protein diversity in organisms.The ferritin heavy polypeptide-like 27(fthl27)gene is one of the members of the ferritin gene family,and the ferritin gene plays an important role in the iron metabolism,iron storage,inflammatory reaction,and the maintenance of normal cell growth and autoimmunity,which also makes the exploration of various aspects of the ferritin gene function become a hot research topic nowadays.In order to adapt to the environment and promote their survival in the process of evolution,organisms have gradually formed a perfect anti-infection mechanism the immune system,which is divided into innate immunity and adaptive immunity.Fish is not only the vertebrate with the largest species and quantity in the biological world,but also has an immune defense system similar to that of higher vertebrates.However,although the mechanisms of innate immunity and adaptive immunity of fish are also quite perfect,compared with higher vertebrates,the specific immune response function of fish is still too low,and the ability to resist the invasion of foreign organisms is also weak.Therefore,fish mainly rely on innate immunity in resisting the invasion of foreign microorganisms,which also leads to fish becoming a model species in the study of innate immunity.At this stage,in the study of ferritin gene,there are few studies on the effect of variable splicing on its regulatory function,and there are also few reports on the study of variable splicing and ferritin gene on fish innate immunity.Miichthys miiuy is a kind of bony fish,which is a common economic fish in China’s coastal areas and has high nutritional value.However,the development of aquaculture is greatly affected by pathogenic microorganisms,so it is particularly important to understand the mechanism of fish immune response.Since the laboratory research on the gene of Miichthys miiuy and innate immune regulation has become mature,this study uses Miichthys miiuy as the test material.Through the analysis and comparison of its transcript library,it is found that there is a variable shear event on ferritin gene fthl27 of Miichthys miiuy.Therefore,this experiment has identified and analyzed the bioinformatics of the variable shear of ferritin gene fthl27 of Miichthys miiuy,At the same time,we also explored the regulatory role of this gene and its expression in fish innate immunity.The main results of this study are as follows:1.Cloning and bioinformatics analysis of the variable fragment of ferritin gene fthl27 of Miichthys miiuy.Based on the second and third generation sequencing,the analysis and comparison of Miichthys miiuy transcript library found that there was a variable splicing event in the ferritin gene fthl27 of Miichthys miiuy,and the sequence information of the fthl27 gene of Miichthys miiuy was obtained by comparing transcriptome library of Miichthys miiuy with other homologous fish gene library,thus the sequence information of the three variable splices of the fthl27 gene of Miichthys miiuy was obtained through PCR and sequencing technology,and it was named as fthl27,fthl27-α and fthl27-β,and also cloned their eukaryotic expression plasmids.Based on this,through bioinformatics analysis software,immunofluorescence,immunoprecipitation and other experimental techniques,in-depth analysis of three variable splices of the fthl27 gene in Miichthys miiuy.It was found that the fthl27 gene of Miichthys miiuy underwent variable splicing,resulting in three different splicing subtypes,with the sequence size of 531 bp,252 bp,and 315 bp respectively;The gene is highly conserved in teleost fish,and has close genetic relationship with Pseudosciaena crocea;The expression of three spliceosomes was higher in the swim bladder cells and eye cells of Miichthys miiuy,while the spliceosome fthl27-β in spleen cells,renal cells expressed very little.2.The three variable splices of the fthl27 gene in Miichthys miiuy are involved in the inflammatory signaling pathway and antiviral immune response.The expression profile of three kinds of variable splices of the fthl27 gene in Miichthys miiuy after LPS and ploy(I:C)stimulation was analyzed by double luciferase activity detection and fluorescence quantitative PCR detection.The results showed that the expression of three kinds of variable splices of the fthl27 gene in Miichthys miiuy increased with time,suggesting that their expression is associated with inflammation and antiviral immune response.Then through the cytokine test of inflammation and antiviral immune response,the results further confirmed that it participated in the inflammatory signal pathway and promoted it.In the antiviral immune response,fthl27 and fthl27-α plays a promoting inhibitory role,while another shear body,fthl27-β has a promoting effect.To sum up,this study found that there are three variable splices of ferritin gene fthl27 of Miichthys miiuy and identified them.Secondly,it has been studied on the innate immunity of Miichthys miiuy.It has been found that three variable splices participate in and promote the inflammatory reaction.In terms of antiviral immunity,fthl27 and fthl27-α plays an inhibitory role,while the shear body fthl27-β contrary.This study not only confirmed the existence of variable splicing events in ferritin genes and the involvement of ferritin genes in the regulation of innate immunity,but also confirmed that variable splicing led to the diversification of gene functions,and provided some support for the research of signal pathways in fish innate immune response,and also enriched the regulatory network of fish innate immunity.