Efficient Gene Expression System in Medaka Embryos Enables Functional Characterization of nt5c1a Paralogs Involved in Inosine Monophosphate Metabolism.

Optimizing transient expression systems in fish embryos is crucial for rapid gene function analysis. Here, we established an efficient system in medaka (Oryzias latipes) embryos by evaluating nucleic acid type and injection site. Our results revealed that injecting the elongation factor 1αA (ef1αA) promoter-driven plasmid into the yolk yielded the highest expression on day 1 post-fertilization. Using this optimized system, we investigated cytosolic 5'-nucleotidase 1a (nt5c1a), which is involved in the metabolism of inosine monophosphate (IMP), an umami flavor compound. In silico analysis revealed that medaka had two nt5c1a paralogs: nt5c1aa and nt5c1ab. While nt5c1ab retains conserved substrate-recognition sequences and exhibits significant IMP degradation activity, nt5c1aa has lost these functions. Structural analysis using AlphaFold revealed that the Nt5c1aa L305P mutation causes local conformational changes near the substrate-binding site, potentially altering substrate orientation without disrupting the overall protein fold. Our expression system demonstrated that this single L305P substitution partially restored IMP-degrading activity in Nt5c1aa, confirming that residue 305 is a key determinant of its functional divergence. Our findings provide a robust foundation for molecular breeding to enhance umami flavor in farmed fish. Specifically, the targeted manipulation of these nt5c1a paralogs could facilitate developing breeds with maximized IMP accumulation in muscle tissues.