| The great blue-spotted mudskipper(Boleophthalmus pectinirostris)is an amphibian fish that can adapt to different temperatures and salinities and live on mudflats in intertidal zones and mangroves,feeding on benthic algae and organic debris.When exposed to air,the species has the ability to breathe through the skin.This species is often distributed along the Northwest Pacific coast,in Korea,Japan,China,Vietnam and Malaysia,and in the main coastal beaches of China,Jiangsu,Zhejiang,Fujian,Taiwan,Guangdong and Guangxi provinces.In China,over the past 30 years,the population of the great blue-spotted mudskipper has declined sharply due to overfishing,fragmentation of natural habitats or environmental damage.At present,there are few studies on all aspects of the great blue-spotted mudskipper.In order to better protect the continuity of B.pectinirostris,we have studied the immune genes of B.pectinirostris.By studying the immune genes of B.pectinirostris,we can better understand the mechanism of B.pectinirostris adapting to external environmental pressure,and provide a reference for the following study of B.pectinirostris.In this study,we cloned and analyzed the coding and non-coding regions of IL-1? and IL-8 genes of B.pectinirostris,and analyzed their gene and amino acid sequences.Through the establishment of phylogenetic trees of two genes,the evolutionary analysis of these two genes was carried out,and their evolutionary patterns were explored.In this study,we also carried out the experiments of Vibrio anguillarum infection and poly(I:C)injection to explore the expression patterns of IL-1?,IL-8 and IL-10 genes in the fish stimulated by different pathogens.The main results are as follows:The coding region and part of non-coding region of IL-1? and IL-8 genes were cloned successfully in this experiment.The analysis showed that the CDs region of IL-1? gene was 720 BP in length,encoding a polypeptide consisting of 239 amino acids.The non-coding region contained three ATTTA motifs.The predicted amino acid sequence contained a precursor peptide of 51 amino acids and a 171 amino acid in length.In the domain 201-221 of amino acid sequence,there is a signal sequence of IL-1 gene family,which consists of 21 amino acids.The coding region of IL-8 gene is 306 BP in length.It encodes a polypeptide chain composed of 101 amino acids.As shown in Figure 2,the first 18 amino acid residues are signal peptide sequences(MKLCVAVMLGTLFVLANGM).After secretion,the mature peptide is formed by cleavage between methionine and serine as shown in Figure 1.The peptide chain domain SCY encoded by IL-8 gene consists of 62 amino acids.In this paper,phylogenetic tree was established to analyze the evolution of IL-1? and IL-8 genes in B.pectinirostris.The analysis showed that IL-1? gene was positively selected in the evolutionary process.Fifteen positive selection sites were found in the evolutionary branch model.Through the analysis of the evolutionary branch-site model,it was inferred that IL-1? gene was positively selected at least three times in the evolutionary process,respectively.It occurs in the early evolution of vertebrates,in the ancestral branches of birds and in the ancestral branches of mammals.Evolution analysis of IL-8 gene shows that IL-8 gene has been highly conserved by purified selection in the evolutionary process,which indicates that IL-8 gene is stable in the evolutionary process and has been in an irreplaceable position.The expression differences of IL-1? and IL-8 genes in different healthy tissues were quantitatively analyzed by fluorescence.The results showed that although IL-1? and IL-8 genes were expressed in different tissues,the expression levels of IL-1? and IL-8 genes were quite different.The expression of IL-1? was highest in the spleen,followed by gill,while the expression of IL-8 gene was highest in brain,spleen and gill,and lowest in skin.It was speculated that the expression level and expression pattern of immune genes in different tissues were different in different fishes.The expression patterns of IL-1? and IL-10 genes in liver and spleen,IL-8 gene in liver,spleen and brain infected by V.anguillarum and poly(I:C)were analyzed by fluorescence quantitative experiment.The results showed that:(1)IL-1? gene of B.pectinirostris was more sensitive to virus infection,and different fish spleens might react differently to virus infection.(2)IL-8 gene is very important for the immune response of liver and spleen after viral and bacterial infection,and the immune response of spleen is earlier than that of liver.Brain damage may occur in the brain tissue of B.pectinirostris under the stress of bacteria and viruses,and the production of IL-8 may come not only from macrophages,but also from glial cells.(3)For IL-10 gene,it may have different activation pathways in liver and spleen after V.anguillarum infection. |
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