Cloning, Expression Of Genes Encoding New Trehalose-producing Enzyme And Trehalose-hydrolyzing Enzyme From Nostoc Flaglliforme

Trehalose is an inducible component in the organisms. It plays great role in protection and stabilization of biological structures such as cell membrane, protein and nucleic acid under varies stress condition and significantly enhance organism's resistance. Nostoc flaglliforme which distributed in west and northwest of China widely is one of valuable terrigenous cyanobacterias. Its'living envioroment is extremely atrocious. To study the metabolic mechanism of trehalose in Nostoc flaglliforme, We cloned two putative thehalose genes (TreY and TreH) DNA fragment from Nostoc flaglliforme and express these putative genes in E. coli. We proved one of the purified recombinant proteins (TreH) do behaves as trehalose hydrolysis function in vitro. This result filled up the blank of trehalose metabolism study in Nostoc flaglliforme.The main contents and results of this experiment are as follows:1.To clone the genes, higher homologous regions were detected to be amplified with degenerate PCR through the procedure Align of clustalx(1.83).exe.2.Extracted total DNA from Nostoc flaglliforme. Used primers to amplify the putative TreY and TreH gene from total DNA with PCR method, then clone them into pGEM^?-T Easy vector to construct the vector pGEM^?-T-TreY and pGEM^?-T-TreH for sequencing of these genes.3.Used bioinformatic methods to forecast the ORF of TreY and TreH, the homology , primary structure, secondary structure, tertiary structure of coding protein, structural domain, and physical and chemical property .4.Constructed expressing vector pET32b-TreY and pET32b-TreH with restriction enzyme cutting sites of BamHI and SalI for expression in E. coli.5.Used IPTG to sucessfully induce TreY and TreH to express in E. coli. The molecular weights of the recombinant TreY and TreH protein are as the same as forecasted, and they are exist as inclusion bodies in E. coli.6.After denatured and refolded the TreH inclusion bodies,the refolded protein showed low activity of trehalose hydrolysis maybe due to the exist of other proteins in inclusion bodies,further purified the refolded TreH protein through Ni⁺ column according to the his-tag structure in vector pET-32b, and the pure TreH protein showed trehalose hydrolysis activity, 82.6U/mg protein.The results showed that the TreY and TreH genes were successfully cloned from Nostoc flaglliforme, and expressed in E. coli, the putative TreH do behave as trehalose hydrolysis activity in vitro, these results provided foundation for further research.