| Among all the genes in veterbtates,miRNAs account for 1-3 percent,which nearly partispate in all the physiological processes.MiRNAs possess high conservation,time sequence and tissue specificity,these characteristics suggest the specificity and complexity of miRNAs in regulating biological function.Previous studies have shown that about 30 percent human genes appear to be conserved miRNA targets,and these genes are involved in all kinds of physiological process,including disease,cell differentiation,tissue development,apoptosis and reproductive development.One miRNA could regulate more than one gene,and one gene also can be regulated by many miRNAs.Although numerous miRNAs have been found,only a small part have been well researched in function and mechanism.It is urgently to determine the target genes of miRNAs.Medaka(Oryzias latipes)is a small size freshwater fish with many advantages,such as transparent embryos,short life cycle and it possess many mutants.In addition,medaka has sex chromosome on which DMY gene is located that is required for male sex determination,and foxl3 is required for female sex determination.Medaka has complete genome sequences and a large number of EST expression library.Furthermore,medaka has a lot of well established cell lines,including diploid embryonic stem cell lines,haploid stem cells,adult germline stem cells and neural stem cell lines from the eyes.Recently,gene editing techniques,including Morpholino(MO),ZFN,TALENs,and CRISP/Cas9 have been established in medaka.We have made long-term efforts to understand germ cell development and sex regulation related genes in medaka,such as dazl,dnd,piwi and vasa.All of these showed that medaka is a good model for development biology and stem cell study,and more and more scientists are using medaka as model in the world.The results would benefit functional research of miRNAs involved in reproduction and sexual differentiation in medaka.Up to now,many miRNAs have been identified in medaka,such as miR-430,miR-202-5p,miR-26 a,miR-210,the related studies in medaka are still lacked.In this study,we screened two miRNAs related to gonad development,predicted and identified the target genes,tried to understand the potential function of these miRNAs and target genes in medaka gonads.It was the first time to identify the target genes of miR-202-5p and miR-26 a by injection in medaka adult,and analysis the target genes(ddx3x & ddx3y)expression pattern in medaka.The mRNA level expression of ddx3 x and ddx3 y was downregulated or up-regulated obvilously after agomir(antagomir)injection,and different doses could strengthen or lighten expression trend.Dual luciferase report system verified the target site in structure.MiR-202-5p and miR-26 a are expressed during the whole embryo stages,previous study showed that in adult gonads of medaka,miR-202-5p could be detected in germ cells,and in this study,we found that both ddx3 x and ddx3 y are expressed in germ cells,other studies verified the same results in different organism.The cDNA of ddx3 x contains 2031 bp nucleotides,encoding 676 amino acids.The cDNA of ddx3 y contains 2298 bp nucleotides,encoding 765 amino acids.Amino acid alignment revealed that both ddx3 x and ddx3 y have a conserved DEAD-box motif,ATP binding motif and RNA binding motif.RT-PCR indicated the expression of ddx3 x and ddx3 y mRNA in different tissues and developing embryos.In the ovary,ddx3 x was dectectd in early stages of germ cells,and in the testis,ddx3 x was detected in spermatids and sperms,which is opposite to ddx3 y in the testis.Based on the results,we predict that ddx3 x and ddx3 y may have potential function in the regulation of gonad development and reproduction in different period,and it also provide a basis for further research in the development and reproduction of medaka. |
