Study On The Biological Function Of CPRL In C.elegans By Using RNAi Technique

The cPRL is one of tyrosine phosphatases in C.elegans and belongs to the subfamily of the tyrosine phosphatase super-family. The tyrosine phosphatase super-family consists of many diverse members of enzymes that play an important role in various cellular processes. Some of the enzymes are specific for phosphotyrosyl residues while others can act on both Ser/Thr and Tyr. Protein phosphorylation is controlled by the coordinate action of protein kinases and phosphatases and has a crucial role in cell proliferation, differentiation, and transformation. In this study, we used C. elegans as a model system to study the cPRL enzymes. C. elegans was initially introduced as a model system for biological research in 1965 by Sydney Brenner, for which he was awarded the Nobel Prize in Physiology and Medicine in 2002. C.elegans is particularly easy and economical to maintain in the laboratory. Growth of C. elegans is rapid. The entire life-cycle, from an egg to an adult producing more eggs, takes just 3.5 days at 20oC. In recent years, some of reports have discovered that knockdown some of special enzymes by RNA interference (RNAi) has profound effects on the behaviors in C.elegans worms. The purpose of the reports is to show that the special enzymes have important biological functions. In our study, we will discovered some characterize of cPRL in the biochemical and biological functions. This study is aimed to characterize biochemical functions of the cPRL enzymes, to reveal the mechanism by which the cPRL enzymes are regulated, and to define the correlation between their biochemical activities and biological functions in C. elegans. RNAi has become the dominant reverse genetics method in C. elegans,where gene silencing can be initiated by injecting dsRNA,soaking worm in dsRNA-containing solutions,or feeding worms dsRNA-producing E.coli.We focused on the use of feeding-based RNAi. This method is simple and highly consistent. RNAi phenotypes induced with this method can be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNA. We choose the pPD129.36 plasmid to make expressing vector for express dsRNA. The vector has a bi-directional promote,which will synthesis both sense and anti-sense dsRNA by IPTG induce . we used the pPD129.36 vector to carry desired cDNA inserts,and to amplify and expression it in HT115(DE3). HT115 carries an rnc null mutation. Rnc encodes a dsRNA-specific endonuclease (RNaseIII), an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. In addition, the E. coli strain also contains the (DE3 lysogen, which enables transcription of genes under control of the T7 promoter. RNAi works in a two-step process. First, the dsRNA is cleaved to yield small fragments siRNAs. Then, the siRNAs target the corresponding mRNA for destruction in an absolutely sequence- specific manner.In worms, only a few molecules of dsRNA per cell are required to silence thousands of target mRNA molecules. Our research came to these results thereinafter:1. We made use of PCR to expand the gene order of cPRL and express relevant dsRNA. We made use of PCR techniques to expand the gene order of cPRL and the result of verification is correct. We construct cloning vector of cPRLs, gene－pBluescript II KS-cPRL. The cutting result of verification by enzymes is ideal. Through recombinating and screening E. coli DH5α, and we screened the positive cloning by LB+Amp. We got target gene from cloning the vector. Through cuting out the pPD129.36 expressing vertor by EcoR I and HindⅢ ,and connecting the genes on it ,we prove that electrophoresis verification of result is correct. We transform expressing vertor into E. coli HT115 and expressing by IPTG. After expressing,we inoculate the E. coli HT115 on RNAi plate for feeding the C.elegans. Verifying cPRL expressing through Western blot, we found the result is correct. 2. The sub-type vari...