Preliminary Study Of Allantoicase Function In The Multi-cellular Development Of Dictyostelium Discoideum

Dictyostelium discoideum is a unicellular eukaryote which phagocytosis bacteria or yeast for food.Under favorable condition,it multiplies as a unicellular erkaryote. Upon starvation,a pathway involves aggregation,mound,slug and fruiting body to form a multi-cellular organism.The progress begins in the signal of concentration gradient of cAMP and accomplishes in the participation of several adhesion molecules and signal molecules.Like the development of metazoan embryonic,multi-cellular development of dictyostelium undergoes a series of morphogenesis and cell differentiation.Despite the multi-cellular development process is relatively simple, the regulatory signaling pathways are as complex as those in metazoan development. So Dictyostelium discoideum has been used as a model organism for researching regulation of signaling molecules during multi-cellular development and cell apoptosis.Gp150 is a membrane glycoprotein,it is encoded by lagC gene.High concentrations of gp150 are present at cell-cell contacts,they regulate cell-cell adhesion by heterophilic interactions.Knock out of lagC gene of wide type strain KAx-3 can generates the mutant type strain AK127.The mutant type strain AK127 can't complete multi-cellular development and the development is arrested at the loose aggregate stage.The obtained data suggests gp150 proteins plays an important role in Dictyostelium discoideum development,it may take part in the signaling pathway for regulating cell differentiation and cell apoptosis.But we don't know all about the signaling pathway yet. When studying gp150,members of my laboratory found a gene fragment relating to the regulation of dictyostelium development(GeneBank number:AY894718).By analysis of BLAST,we knew the fragment was part of allantoicase gene.Allantoicase 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.Ammonia may regulate the differentiation of dictyostelium by suppressing DIF,so it may be an important signal molecule in this progress.But we have not known the details.RNAi is an important means to study these questions.It is a posttranscriptional gene silencing method initiated by double-stranded RNAs,which are homologous to the suppressed gene.In mammals,dsRNAs of less than 30nt were used to study RNAi. But in several non-mammals,it was thought that long dsRNA would be more feasible, because long dsRNA was more effective on suppression.But it was difficult to construct vectors of long dsRNA.We had an exploration of the construction of long dsRNA expression vector,and got satisfied results.Total RNA from KAx-3 cells(developed for 14h)was isolated. After the reverse transcription and PCR reaction,two DNA fragments of about 400bp and 600bp were generated.The fragments were recovered and cloned into pMD18-T vector.After sequencing,we knew that two fragments were 397bp and 642bp respectively.Two fragments shared the same initiation site,and the shorter fragment had the same sequence with the front of the longer fragment.The fragments were then cloned into pAct15Gal vector in opposite directions to generate the RNAi vector against allantoicase gene.Dictyostelium discoideum was grown in HL5,which is axenic and liquid.The RNAi vectors were transformed into Dictyostelium cells by calcium phosphate precipitation or electroporation.Positive cells were selected by the drug of G418.Positive clones could germinate in the plate of G418 resistance 7-12 days later,and the clones were some dots like colony.We could find that the modality of the clones were as same as the modality of Dictyostelium mound under inverted microscope.The modality of clones would not change from then on,and they could not form the fruiting bodies.We can speculate that the silence of allantoicase gene by RNAi results in the breakage of Dictyostelium development signal nets.So the Dictyostelium can not form fruiting bodies.So allantoicase plays an important role in the signal conduction of Dictyostelium.However,we need further experiments to confirm the approach and the protein molecules.This research lays the foundation for the study of allantoicase gene function and its role in the signal conduction of Dictyostelium further.And it also contribute to the study of the whole signal pathway of Dictyostelium.