Effect Identification Of RNAi Expression Vector Targeting Allantoicase Gene In Dictyostelium Discoideum

The social amoeba Dictyostelium discoideum which phagoeytosis soil bacteria for food is a unieellular eukaryote.Under favorable condition,it multiplies as a unicellular organism.UP on starvation,a pathway involving aggregation,mound,slug, eulmination stages induces the formation of a fruitingbody consisting of a head of spores supported on a stalk of vacuolated cells.Spores await dispersal and germinate in favourable conditions for amoeboid growth.Cells of Dictyostelium discoideum are triggered to undergo development when they have depleted their food souree.During the early hours of development,cell adhesion molecules play an important role in maintaining the integrity of cell aggregation and influence subsequent cell-type differentiation and cell sorting.gp150 is normally first expressed at the loss aggregation stage and this adhesion moleculeis important for development.The membrane glycoprotein gp150 encoded by Lag C,play a role in cell-type specification by heterophilic interactions.A strain in which lagC is disrupted,AK127,undergoes normal chemotaxis,but is arrested at the loose aggregate stage.So we inferred that gp150 play a key role during the whole development of dictyostelium.It may be in the signal transduction way of cell differentiation and apoptosis.However the concrete signal route has not been known.To study the influence of the cell adhesion molecule gp150 on the expression of genes needing for Dictyostelium discoideum development,mRNA differential display was used to analyse the differences of gene expression between the wild strain KAx-3 cells and the mutant strain AK127 cells.Members of my laboratory found a gene fragnlent relating to the regulation of Dictyostelium discoideum development (GeneBanknumber:AY894718).By analysis of BLAST,we knew the fraglnent was part of allantoicase gene.Allantoiease is an important enzyme in purine degradation. In primates,birds,and insects,purine is converted into uric acid and then excreted.But in microorganisms,amphibians,and fish,uric acid is converted into allantioc acid. Allantoicases catalyze the hydrolytic conversion of allantoic acid into ureidoglycolate and urea.Ureidoglycolate is converted into ammonia,carbon dioxide,and glyoxylate. It was reported that ammonia inhibits stalk cell differentiation and promotes differentiation of pre-spore cells to spores by suppressing DIF cumulation.Ammonia may be an important signal molecule which regulate the development of Dictyostelium. However the concrete regulation has not been known.So we transfected RNAi expression vector pAct15Gal-allCi into KAx-3 as to study the gene function in Dictyostelium development.The RNAi expression vector pAct15Gal-allCi was transfected into KAx-3 by electroporation.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 from72.18%to 0.67%after RNAi transfection.The results suggest that allantoicase is interfered,which is required for the development of Dictyostelium discoideum.