大乳头水螅原癌基因c-Myc的克隆、生物信息学分析及原核表达生物化学与分子生物学专业论文.docx

IV IV Gene cloning, bioinformatic analysis and prokaryotic expression of protooncogene c-Myc in Hydra magnipapillata Abstract Protooncogenes are normal genes involved in cell growth, cell division and cell differentiation, but their mutations can make themselves become oncogenes. So far, more than one hundred species of protooncogenes was found. Nucleic acid transcription factor c-Myc is one of the famous protooncogenes, because it could be the causative agents of many cancers. It was found that hydra as a primitive diploblastic animal has c-Myc gene by use of theoretical analyses about DNA sequences from hydra genome project completed in 2010. Can the protooncogene c-Myc of hydra be transformed into a oncogene? what is the exact physiological function of c-Myc in hydra? is the expression of c-Myc in hydra related to the strong regenerative capacity of hydra? these problems need to be answered urgently. In view of this, we cloned the c-Myc gene in Hydra magnipapillata and carried out the following research work: A SMART RACE cDNA library of H. magnipapillata was constructed successfully, and RT-PCR method was used to clone c-Myc gene cDNA sequence of H. magnipapillata. The coding region’s full-length cDNA of c-Myc gene in H. magnipapillata was 945 bp, encoding for 314 amino acids, and the predicted c-Myc protein molecule consists of one transcriptional activation domain and one helix loop helix (HLH) domain. It will built the foundation for the further study about function and evolution of c-Myc protein and the origin of c-Myc gene that c-Myc gene cDNA sequence of H. magnipapillata was successfully cloned. Based on the cDNA sequence of H. magnipapillata c-Myc gene and the characters of multiple cloning sites of prokaryotic plasmid expression pET-LG, a pair of primers were designed. The cDNA sequence of c-Myc gene with special restriction endonuclease sites was synthesized by use of PCR, and PCR product was double-digested by BamH I and EcoR I before being inserted