Isolation and characterization of a PUF-domain of pumilio gene from silkworm <i>Bombyx mori</i>

Abstract

Pumilio is a sequence-specific RNA-binding protein that binds to target mRNA to repress its translation. The PUF-domain, the RNA-binding motif of pumilio, is highly conserved across species. In the present
study, a partial pumilio gene with complete PUF-domain in Bombyx mori has been cloned using 3’ and 5’ RACE for the first time, designated as BmPUM. The sequence of BmPUM has been registered in
GenBank under the accession number FJ461590. Comparative sequence analysis revealed that the deduced protein BmPUM contains a PUF-domain and shares 83% identity with Drosophila pumilio,
hence belongs to pumilio family. One encoding sequence of BmPUM fragment was successfully expressed in Escherichia coli. Western blotting indicated that the anti-BmPUM antibody and anti-
Drosophila pumilio antibody all could specifically detect BmPUM expressed in prokaryotic cells. The tissue expression pattern performed by real-time PCR and western blotting demonstrated that BmPUM
expressed in various tissues, especially in testis and ovary. Those data collectively indicated that BmPUM belongs to the extremely high conserved RNA-binding domain. Conserved function of pumilio
protein in invertebrates and vertebrates suggested that BmPUM could also play an important role in the proliferation of silkworm germline stem cell.