MiRNA Expression Profiles And Expression Of SOD Gene In Gill Of Eel Almond Under Different Salinity

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate gene expression by post-transcriptional repression of mRNAs. Recently, several miRNAs have been confirmed to take part in osmoregulation in fish. However, superoxide dismutases (SODs), as the primary antioxidant enzymes, play a first role in the process of preventing oxidative stress caused by excessive superoxide anion (O2-) in living organisms. In the studing of catadromous fish, there are still no reported that SOD actually participate in osmoregulation. Based on the Illumina sequencing data and expression patterms of SOD genes under different salinities and injection of Aeromonas hydrophila, we aimed to probe the osmoregulatory machnism in Anguilla marmorata and reveal the expression patterms of SOD genes. Our study can provide foundation of antioxidant regulatory mechanism in further researches.(1) MiRNA expression analysis of gill tissue in A. marmorata under different salinities.With the assistance of Illumina Hiseq 2000 platform, the different salinities treated (FW, salinity of 0; BW, salinity of 10; SW, salinity of 25) sRNA of gill tissue of A. marmorata was sequenced. After quality control, totally 11,339,168,11,958,406 and 12,568,964 clear reads were obtained from 3 different libraries, respectively. Meanwhile,34 conserved miRNAs and 613 novel miRNAs were identified and predicted by using sequence data. MiR-10b-5p, miR-181a, miR-26a-5p, miR-30d and miR-99a-5p were dominantly expressed at three salinities in our study. Totally,29 miRNAs were significantly up-regulated, while 72 miRNAs were significantly down-regulated in brackish water (10‰ salinity) compared with fresh water (0%o salinity); 24 miRNAs were significantly up-regulated, while 54 miRNAs were significantly down-regulated in sea water (25%o salinity) compared with fresh water. Similarly,24 miRNAs were significantly up-regulated, while 45 miRNAs were significantly down-regulated in sea water compared with brackish water. The expression patterns of 12 dominantly expressed miRNAs were analyzed at different time points when the eels transferred from fresh water to brackish water or to sea water. Interestingly, miR-122, miR-140-3p and miR-10b-5p demonstrated special osmoregulatory effects in certain salinities. In addition, the identification and characterization of differentially expressed miRNAs at different salinities can clarify the osmoregulatory roles of miRNAs, which will shed lights for future studies on osmoregulation in fish.(2) Preliminary research of SOD gene of A. marmorataTwo distinct members of SOD family from the marbled eel A. marmorata (denoted as AmMnSOD and AmCu/ZnSOD) were identified by using homology cloning and rapid amplification of cDNA ends (RACE). The structural analysis revealed common features of SOD family in both AmSODs from primary to tertiary levels, including two MnSOD signature motifs and two Cu/ZnSOD signature motifs in AmMnSOD and AmCu/ZnSOD, respectively. Phylogenic analysis indicated that MnSOD and Cu/ZnSOD proteins were both within fish monophyletic clade. The tissue distribution analysis demonstrated that AmMnSOD and AmCu/ZnSOD were expressed at higher levels in muscles and livers.We analysed the mRNA, Ezymatic activities and protein expression patterns of two AmSOD genes under different salinities treatment in five distinct tissues (gill, kidney, intestine, muscle and liver) of A. marmorata. All the results indicated that gene expression and enzyme activities of the two AmSODs in juvenile marbled eels could be effectively enhanced by low salinity but inhibited when the salinity was higher than the body tolerance.The time-course dynamic expressions of AmSODs were observed through the experimental challenges with A. hydrophila, which were all significantly altered in the three distinct tissues (gill, kidney and liver). Results of the enzyme activity and western blot analysis also showed that the dynamic changes of AmSODs which were significantly regulated by A. hydrophila challenge (P< 0.05). All the result showd liver and kidney tissues had more sensitive immune response compare with gill tissue of A. marmorata. And our study indicated the significant role of the two AmSOD genes in initate immune response of A. marmorata.