| Grass carp(Ctenopharyngodon idella)is the species with the highest aquaculture production in the world.Grass carp farming is susceptible to disease,and improving the disease resistance of grass carp has become one of the important means to improve the survival rate.The disease resistance of fish is related to genetic factors.Gadd45 family genes are an important class of DNA damage repair genes,which have the functions of regulating cell cycle,participating in apoptosis and immunity.They are found in grass carp with five members,namely Gadd45 aa,Gadd45ab,Gadd45 ba,Gadd45bb and Gadd45 g.In this paper,we selected five genes of the Gadd45 family from the diseaseresistant transcriptome database and cloned three of them,and screened five miRNAs corresponding to the five members of the Gadd45 family of genes(miR-731,miR-23a-3p,miR-23a-5p,miR-148 and miR-429b),studied their structure and function.The main research results of the paper are as follows:1.The accession numbers of the three genes such as Gadd45 aa,Gadd45ab and Gadd45 g are KX523186,KX523187 and MK829489,the open reading frames are 474 bp,480 bp and 480 bp,respectively,and the encoded amino acids are 157 aa,159 aa and 159 aa,The molecular weights are 17,547 Da,17,708 Da and 17,720 Da,respectively,and the theoretical isoelectric points are 4.98,4.45 and 4.03,respectively.It is predicted from the SMART website that grass carp Gadd45 aa,Gadd45ab and Gadd45 g have ribosomal protein L7 Ae domains of 90 aa,106 aa and 91 aa,respectively.Grass carp Gadd45 aa,Gadd45ab and Gadd45 g were expressed in 12 healthy tissues of grass carp and 6 immune-related tissues after Aeromonas hydrophila infection.After processing three genes by means of overexpression and interference,all three genes can activate the MAPK signaling pathway and upregulate the immune genes il-8,ifn and tnf-a.2.Predicted by the target gene,it is known that grass carp Gadd45 aa is the target gene of miR-731.After infecting CIK cells with Aeromonas hydrophila,the expression patterns of miR-731 and grass carp Gadd45 aa were simultaneously detected at different time points.The results showed that the expression trends of the two were opposite at different time points.miR-731 overexpression / interference can inhibit the expression of Gadd45 aa in grass carp.The dual luciferase reporter gene results showed that the luciferase activity of the wild-type(WT)miR-731 mimic was reduced,while the miR-731 mutant(Mut)was not affected,proving that the grass carp Gadd45 aa was the target gene of miR-731.In CIK cells,overexpression of miR-731 can significantly inhibit the expression of 6 immune-related genes(p38,jnk,erk,tnf-a,ifn and il-8),and miR-731 inhibitor significantly increased the expression of six genes expression.Gadd45 aa and miR-731 can induce CIK cell apoptosis after overexpression.In summary,miR-731 can regulate the expression of immune genes by targeting Gadd45 aa,but may regulate its target genes to participate in apoptosis.3.To identify that Gadd45 ab was the target gene of miR-23a-3p and miR-23a-5p,transfect CIK cells with miR-23a-3p and miR-23a-5p mimics,and q PCR detected that the expression of Gadd45 ab was significantly suppressed.In contrast,Gadd45 ab expression was significantly increased in cells transfected with miR-23a-3p and miR-23a-5p inhibitors.These results indicate that miR-23a-3p and miR-23a-5p jointly regulate the target gene Gadd45 ab.The dual luciferase reporter vector experiment further confirmed that Gadd45 ab is the target gene of miR-23a-3p and miR-23a-5p.Overexpression of miR-23a-3p significantly suppressed the expression of jnk,erk,tnf-a and ifn,and miR-23a-3p inhibitor significantly increased the expression of p38,erk,tnf-a and ifn.Mi R-23a-5p overexpression significantly inhibited the expression of p38,jnk and erk,tnf-a and il-8,and miR-23a-5p inhibitor significantly increased the expression of p38,erk,tnf-a and ifn.After infection of miRNA inhibitors with Aeromonas hydrophila,the miRNA expression levels were significantly reduced.According to the caspase 3/7 experiment,it is concluded that miRNA has an inhibitory effect on apoptosis,and its target gene Gadd45 ab can promote apoptosis.Therefore,in grass carp,both miR-23a-5p and miR-23a-3p can activate the expression of immune-related genes and inhibit cell apoptosis by regulating their target genes.4.Dual luciferase reporter gene experiment and miRNA-148 mimics / inhibitors reverse regulation of target genes,verify that Gadd45 ba and Gadd45 bb are both miR-148 target genes.At different time points when Aeromonas hydrophila infected CIK cells,the expression level of miR-148 changed significantly,indicating that miR-148 had a certain response to bacterial infection.After miR-148 overexpression,the expression levels of p38,jnk,erk,tnf-a and ifn decreased significantly,while in the miR-148 inhibitor group,the expression levels of p38,jnk,erk,tnf-a and ifn increased significantly.In summary,miR-148 responds to bacterial stimuli in grass carp,and miR-148 regulates immune gene expression by interacting with its target genes.5.Website predicts that Gadd45 g is the target gene of miR-429 b.The overexpression and suppression of miR-429 b have a negative regulatory function on the expression of immune-related genes il-8,ifn and tnf-a.miR-429 b regulates immune genes by suppressing its target genes in grass carp.This article explores the antimicrobial immunity and apoptosis of grass carp through the interaction of miRNA and target genes.The results provide a target gene for studying the genetic mechanism of grass carp's disease resistance traits,and also establish a theoretical data base for the study of miRNA regulating grass carp antibacterial immunity,and provide a theoretical reference for miRNA research in other aquatic animals. |
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