MOLECULAR CLONING AND IDENTIFICATION OF NANOS3 IN BLUNT SNOUT BREAM (MEGALOBRAMA AMBLYCEPHALA)

Nanos3 is one of the components of germ plasm which is generally considered to be the determinant of primordial germ cells (PGCs) in most of the ovipara. The miR430 bind to the 3′untranslated region (UTR) of nanos3 to mediate the degradation of its mRNA in somatic cells, while in PGCs, the Dnd1 binds to the 3′UTR region of nanos3 to protect it from degradation through miR430. Therefore, the 3′UTR of nanos3 was generally used to mediate the specific expression of fluorescent protein in PGCs. In this study, we cloned and characterized the cDNA of the nanos3 homolog in Megalobrama amblycephala (mananos3). Mananos3 is of 1027 bp length including 48 bp 5′UTR, 490 bp 3′UTR and 489 bp opening reading frame. It encodes a peptide with 162aa. By peptide alignment, there was a conserved RNA binding domain with one zinc finger motif. Phylogenetic analysis revealed that Mananos3 has the highest similarity with its homolog in common carp. Semi-quantitative and Real-time qPCR showed that nananos3 was highly maternally expressed. During early embryonic stage, it was high expressed before 1k-cell stage and then gradually decreased. Mananos3 was specifically expressed in ovary among different selected tissues. Both 3′UTR of mananos3 and zebrafish nanos3 (zfnanos3) can mediate the specific expression of green fluorescent protein (EGFP) in PGCs while it appeared that 3′UTR of mananos3 has higher efficiency and specificity. Finally, the alignment of the 3′UTR of mananos3 and zfnanos3 revealed a potential non-classical recognition site (GCACTA) for miR430 that promotes the degradation of mRNA in non-PGCs tissues.