Construction and Effect Identification of RNAi Expression Vector Targeting Allantoicase Gene in Dictyostelium discoideum

Abstract: This study was aimed to limit cell development in Dictyostelium discoideum by RNAi strategy targeting allantoicase gene (allC). In accordance with the allC sequence in Genbank， partial cDNA sequences (642 bp， 397 bp) from Dictyostelium discoideum were amplified by RT-PCR. The gene-specific sequences (642 bp， 397 bp) of allC were linked in the antisense orientations in order to form hairpin RNA. The resulted construction was then inserted into vector pAct15Gal to obtain the RNAi expression vector pAct15Gal-allCi. Positive clones were obtained by stable transfection KAx-3 using pAct15Gal-allCi and G418 screening. Compared with untransfected KAx-3， transfected cells， arrested early stage of mound， could not complete integrative cell development. Western blot analysis showed allantoicase protein in interference sequence transfected cells was significantly down-regulated. The transfection rate was estimated by FACS analysis， indicating the allantoicase expression decreased from 72.18% to 0.67% after RNAi transfection. The results suggest that allantoicase is interfered， which is required for the development of Dictyostelium discoideum.

CSTR: 32200.14.cjcb.2009.03.0017