Cloning of Trehalase Gene from Arabidopsis thaliana, Overexpression in E.coli and Its Functional Activity Assay in vitro

Abstract:

The molecular researches of trehalose biosynthesis and metabolization have been attended because of its important roles in the cyto-physiology process of animal and plant cell. By using RT-PCR (reverse transcription PCR) method, the cDNA sequence of trehalase has been cloned through the RNA extracted from Arabidopsis sprout in this experiment. 1674 bp cDNA identified by sequencing was constructed to pET30a (+) vector and transformed to BL21 of E. coli. The target protein has overexpressed and has been purificated by using Ni-NTA Agarose, and then its activity assay and enzymatic characteristic were studied in vitro. The results indicated that this plant trehalase gene can overexpress in E. coli and the trehalase protein which purificated through Ni-NTA Agarose has higher hydrolysis activity and its suitable reaction temperature is 45℃. The quantity change between glucose and trehalose occurred obviously in the enzymatic reaction as time through GC-MS analysis. This further confirmed that the trehalase gene come from Arabidopsis has expressed functionally in E. coli.