| Portunus trituberculatus is the main marine culture crab in Chin a.Its growth,molting,development and immunity are susceptible to ch anges in salinity of the water environment.Osmoregulation is one of t he important physiological processes for crustaceans to adapt to exter nal salinity changes,among which ion transport across the membrane i n gill epithelial cells is the main pathway of osmoregulation.micro RN A(miRNA)are a class of functionally important non-coding small m olecule RNAs that can regulate the expression of target genes from t he post-transcriptional level and play important functions in biological development,differentiation,and resistance to stress.It has been shown that miRNAs can be involved in the regulation of osmotic pressure i n aquatic animals,but related studies are less reported in crustaceans.T his study aims to investigate the molecular mechanism of osmotic pr essure regulation in P.trituberculatus at the post-transcriptional level:to efficiently discover the miRNAs related to osmotic regulation by hig h-throughput sequencing,to predict the miRNA target genes by bioinfo rmatics methods,and to verify the regulatory relationships between m i RNAs and their target genes at the in vivo and ex vivo levels.The r esults can help to systematically analyze the mechanism of osmoregul ation in P.trituberculatus and provide a theoretical basis for the select ion and breeding of low salt tolerant species.The research in this pap er consists of the following four parts:1.Identification and analysis of miRNAs in gill tissues of P.tritubercul atus under low salinityHigh-throughput sequencing was used to analyze the gill tissues before and after low salinity stress.478 miRNAs were detected,inclu ding 336 known miRNAs and 142 newly predicted miRNAs.The resu lts of differential analysis showed that 163 miRNAs were significantl y differentially expressed after low salinity stress,indicating that they may play a regulatory role in low salt adaptation.Further analysis rev ealed that the differentially expressed miRNAs were mainly concentra ted at 0-12h after low salinity stress,and most of them showed an u p-regulated expression trend.Ten significantly differentially expressed miRNAs were randomly selected for quantitative analysis,and their e xpression trends were found to be consistent with those detected by sequencing methods,indicating that the results of high-throughput seq uencing and analysis of miRNAs in this study were highly credible.T he experiments in this chapter uncovered a batch of differentially exp ressed miRNAs after low salinity stress,which laid the data basis for an in-depth investigation of the function of miRNAs in low salt adap tation in P.trituberculatus.2.Screening and analysis of ion transport-related miRNAs and their ta rget genesA total of 7949 clearly annotated target genes were obtained fro m the above-mentioned miRNAs,including two ion transport genes clo sely related to osmolarity regulation-Na~+/K~+-ATPase and Na~+/H~+-excha nger.miRNA target prediction results showed that the miRNAs that co uld bind their 3’UTRs were miR-200137 and miR-466f-3p,respectively.’UTRs were miR-200137 and miR-466f-3p,respectively.quantitative a nalysis showed that both Na~+/K~+-ATPase and Na~+/H~+-exchanger genes were highly expressed in gill and hepatopancreas tissues,significantly higher than other tissues(P<0.05).Correspondingly,miR-200137 and miRNA-466f-3p were also significantly highly expressed in gill and h epatopancreas tissues,and both had a tendency to co-localize in the ti ssue distribution.Further study revealed that miR-200137 and miR-466f-3p showed a significant negative correlation with the expression tre nds of their potential target genes Na~+/K~+-ATPase and Na~+/H~+-exchang er in gill tissues after low salinity stress(P<0.05).The above results te ntatively demonstrated that miR-200137 and miRNA-466f-3p might be important factors regulating the expression of Na~+/K~+-ATPase and N a~+/H~+-exchanger genes.3.In vitro validation of ion transport-related miRNA-target genesBased on the above findings,we further verified the regulatory re lationships of miR-200137 and miR-466f-3p with their potential target genes,Na~+/K~+-ATPase and Na~+/H~+-exchanger,respectively,at the cellu lar level with the help of dual-luciferase assays.Firstly,we constructed Na~+/K~+-ATPase-3’UTR-wt(mut),Na~+/H~+-exchanger-3’UTR-wt(mut)d ual luciferase expression vectors and luciferase-free NC mimics,miR NA mimics(miR-200137 and miR-466f-3p)expression vectors,and the n transfected 293T cells.The experimental results showed that lucifera se expression was significantly down-regulated only when miR-200137 was cotransfected with Na~+/K~+-ATPase-3’UTR-wt(P<0.001),indicatin g that miR-200137 was able to directly inhibit Na~+/K~+-ATPase gene e xpression at the in vitro cellular level.Similarly,the results of dual-luci ferase assay of miR-466f-3p with Na~+/H~+-exchanger showed that miR-466f-3p was able to directly inhibit the expression of Na~+/H~+-exchang er gene.The results of in vitro experiments showed that the 2 miRN As could directly inhibit the expression of their target genes at the c ellular level.4.In vivo validation of ion transport-related miRNA-target genesBased on the in vitro experiments,we tried to verify the relatio nship between miRNA and its potential target genes again from the i n vivo level.After in vivo injection of miRNA agomir,the expressio n of miR-200137 was continuously upregulated,and the expression p eaked at 48h,528 times that of 0h(P<0.05).Meanwhile,the expressi on and enzyme activity of Na~+/K~+-ATPase gene were significantly inh ibited,with the lowest expression at 48h and 24h,respectively,signif icantly downregulated 25-fold and 3.66-fold compared with 0h(P<0.05).Similarly,injection of miRNA agomir successfully upregulated the e xpression of miRNA-466f-3p,with the highest expression at 24h,which was 299-fold higher than that at 0h(P<0.05).Correspondingly,Na~+/H~+-exchanger was significantly downregulated 19.8-fold after 48h of inje ction compared to 0h(P<0.05).In addition,we also examined the os molarity and ion concentration of hemolymph.The hemolymph osmola rity and Na~+and K~+ion concentrations were found to decrease after miR-200137 and miR-466f-3p agomir injections.The above results de monstrate that miR-200137 and miR-466f-3p overexpression in vivo i nhibits the expression of Na~+/K~+-ATPase and Na~+/H~+-exchanger,thus af fecting their functions in the regulation of osmotic pressure in P.tritu berculatus. |
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