Studies Of The Biological Functions Of Protein Tyrosine Phosphatase 3 In C.elegans By RNAi Technique

The protein tyrosine phosphatase 3 is a subfamily of leukocyte-common antigen related (LAR) and receptor protein tyrosine phosphatases include the leukocyte-common antigen related (LAR) family. RPTPs is a family of transmembrane protease, playing a key role in cell adhension and cell signal transduction. The type IIa family of receptor protein tyrosine phosphatases (RPTPs) include LAR which is a cell adhension molecule, regulating the development of many tissues including the nerve system. The extracellular domains of LAR-like RPTPs contain Ig-like and fibronectin type III (FNIII) repeats. The extracellular domains of LAR contain some cell adhesion proteins, suggesting LAR is related with cell adhesion and the tyrosine phosphorylation in cells.PTP-3 of C.elegans is a kind of high conservative LAR-RPTP. The C. elegans genome contains 26 receptor proteintyrosine phosphatases, but the LAR-RPTP in C.elegans is the only one RPTP, which is PTP-3. The two function domains of PTP-3 in cell are D1and D2, the active center of PTP-3 is D1, which is the catalytic domian, and the D2 is a subsidiary domain which contributes to the catalytic function of D1.There are mainly two isoforms of PTP-3 in C.elegans, PTP-3-A and PTP-3-B, ptp-3, the single C. elegans type IIa RPTP gene, encodes two isoforms which differ in the extracellular domain. PTP-3A is very similar to vertebrate LAR, whereas the shorter isoform, PTP-3B, lacks the Ig domains and the first four FNIII repeats. PTP-3-A and PTP-3-B exist near or in the synapse and the tissues near synapse in C.elegans, and they are responsible for the development of synapse and the neuroblast migration during embryogenesis respectively. The guidance and migration of axon are in progress in two axles, back-abdomen axle and front-back axle. The family of unc gene plays a key role in the back and abdomen axle, but in the front-back axle, the vab-8 does, in the C.elegans the four epithelial cell cords twist with muscle and nerves, so that the nerves can control the stretching of the muscle easily. cPTP-3 expressed in many tissues in the early embryogenesis, and finally localize at the synapse and the junction of epithelial cells.It is unclear that the function and the mechanism of cPTP-3 in the cell signal transduction. To study the function further, we use C.elegans as a model to continue the experiment. The nerve system of C.elegans is mainly responsible for the behaviors and their responses to the surrounding environment. They can sense the temperature, stimulus and many kinds of chemotaxis agent and so on. Consider of its simple nerve system, we use C.elegans as a model to help us understand the behaviors of kinds of animals.In this article, we utilize gene engineering to clone the cPTP-3-2D and cPTP-3-D1 genes from cDNA library of C.elegans then we clone the former into the vector pGex-HisC3 to acquire an expression vector pGex-cPTP-3-2D. By transformating it into the E.coli DE3 Roseata, we get the strain that can highly express cPTP-3-2D. Meanwhile, we purify the fusion protein GST-cPTP-3-2D by preparation electrophoresis. Thus, we finally immunize the rabbit to get anti-cPTP-3 antibodies.The gene engineering strain which expresses cPTP-3-D1 dsRNA was constructed by plasmid L4440 and E.coli HT115. We used it to feed C.elegans and observed the phenotypes, such as lifespan, abnormal death, larvae number and the abnormal phenotype of locomotion and vulva development, we come to some conclusions according to the experiment results as follow: 1. The decrease of cPTP-3 expression is not related with mechanism of shortened lifespan directly.2. cPTP-3 contributes to the correct linkage of motor axon synapses and muscle cells.3. The increase of embryo lethality and the abnormality of ovulation lead to the less number of filial generation.4. The decrease of PTP-3 not only make the vulva stretching interrupted, but also affects the normal stretching of body muscle cell.5. The compensation mechanism has in a sense reduced the influence to the nematodes caused by the decrease of cPTP3.The works above explained the biological function of PTP-3 in C.elegans and contributes as the basis for the further study of cPTP-3 and diseases.