Study On The Biological Functions Of Caenorhabditis Elegans Protein Tyrosine Phosphatase PTP-2 Using RNA Interference

RNA interference (RNAi) has undergone a rapid development as a powerful method to the research of Gene Therapy and the Functional Genomics since it has been discovered by Fire etc. in 1998. It is a technique that degrades the specific mRNAs and depresses the corresponding genes, so RNAi can lead to the post-transcriptional gene silencing. C.elegans is a perfect RNAi carrier with short life-span, it is easy to keep in laboratory. Moreover, its transparent epidermis eases the process of observation. Most importantly, C.elegans is a eucaryotic metazoan whose cells, molecular constructions and signaling pathways are similar with higher animals. All these above unique characteristics make C.elegans an excellent model organism.SHP-2 is a member of protein tyrosine phosphatases (PTPase) family. Large amount of researches show that SHP-2 is correlated with various biological processes, such as cell proliferation, differentiation, migration, apoptosis and so on. ptp-2 is homologous with SHP-2, study showed ptp-2 knocked-out C.elegans is abnomal during oogenesis and vulval development, but the mechanism of PTP-2's action and it's biological functions in other aspects are unclear now, so in our experiment we plan to study the biological functions of PTP-2 using RNAi, and to study the mechanism of its action. Our study can be the first step to further study the mechanism of SHP-2 's action in higher animals.The main procedures of this research are as follows. Firstly, we constructed the ptp-2 gene to the expression vector pGex-2T, and get the GST-PTP-2 fusion protein. Secondly, we purified the GST-PTP-2 fusion protein using preparative electrophoresis, and started the immunoreactions in rabbit with the purified GST-PTP-2, the serum of anti-PTP-2 is prepared for the next procedure. Finally, we analyzed the biological functions of PTP-2 using RNAi. 1. We constructed the engineering bacteria that can express the ptp-2 dsRNA. 2. To feed the worms with above engineering bacteria and to detect the expression of PTP-2 by Western Blot. We find the decreased expression of PTP-2 in C.elegans after RNAi. 3. To compare the physiological phenotypes between the RNAi group and the control group, and to analyze the differences. The phenotypes we observed include brood size, life-span, time interval of defecation and the proportion of unnatural death and so on. We find that RNAi group has shortened life-span, more vulval rupture and wormbag. In brood size and the time interval of defecation there is no obervious differences. From above, we can draw the conclusion that: PTP-2 plays an important role in muscle system in C.elegans, and PTP-2 paticipates the life-span related signaling pathways. In the future, firstly, we may use the specific immunofluorescent agent to confirm the relationship between protein PTP-2 and development of muscle system. Secondly, we will investigate the PTP-2 in cell level as a regulator. Finally, we will find the contribution of PTP-2 in signaling transduction and make clear its mechanism of action.The methods we used in our experiment includes clone and expression of enzyme gene of C.elegans, isolation and purification of protein, preparation of multi-clonal antibody of enzyme, RNAi on enzyme of C.elegans and observation of the phenotype changes in C.elegans after RNAi. There are lots of studies on biological functions of SHP-2 in drosophila, mice and human being, whereas, the studies of PTP-2 in C.elegans are rare both at home and abroad. So we expect to make our efforts to reveal the mechanism of PTP-2 and to develop the drug and therapy of PTP-2 related human diseases.