Gene Cloning, Bioinformatic Analysis And Prokaryotic Expression Of Protooncogene C-Myc In Hydra Magnipapillata

Protooncogenes are normal genes involved in cell growth, cell divisionand cell differentiation, but their mutations can make themselves becomeoncogenes. So far, more than one hundred species of protooncogenes wasfound. Nucleic acid transcription factor c-Myc is one of the famousprotooncogenes, because it could be the causative agents of many cancers. Itwas found that hydra as a primitive diploblastic animal has c-Myc gene by useof theoretical analyses about DNA sequences from hydra genome projectcompleted in2010. Can the protooncogene c-Myc of hydra be transformed intoa oncogene? what is the exact physiological function of c-Myc in hydra? is theexpression of c-Myc in hydra related to the strong regenerative capacity ofhydra? these problems need to be answered urgently. In view of this, we clonedthe c-Myc gene in Hydra magnipapillata and carried out the following researchwork:1. A SMART RACE cDNA library of H. magnipapillata was constructedsuccessfully, and RT-PCR method was used to clone c-Myc gene cDNAsequence of H. magnipapillata. The coding region’s full-length cDNA ofc-Myc gene in H. magnipapillata was945bp, encoding for314amino acids,and the predicted c-Myc protein molecule consists of one transcriptionalactivation domain and one helix loop helix (HLH) domain. It will built thefoundation for the further study about function and evolution of c-Myc proteinand the origin of c-Myc gene that c-Myc gene cDNA sequence of H.magnipapillata was successfully cloned.2. Based on the cDNA sequence of H. magnipapillata c-Myc gene and thecharacters of multiple cloning sites of prokaryotic plasmid expression pET-LG,a pair of primers were designed. The cDNA sequence of c-Myc gene withspecial restriction endonuclease sites was synthesized by use of PCR, and PCRproduct was double-digested by BamH I and EcoR I before being inserted intoprokaryotic plasmid expression of pET-LG to construct prokaryotic plasmidpET-LG-c-Myc. After induction of IPTG, the recombinant c-Myc wasexpressed in E. coli BL21(DE3). SDS-PAGE analysis results indicated thesuccessful expression of c-Myc protein. The expression of recombinant c-Mycprotein help to reveal the physiological function of c-Myc protein.