Cloning And Overexpression Of Myrosinase Gene Tgg1 From Arabidopsis Thaliana And Characterization Of Its Recombinant Protein

Crucifers are widely distributed in nature, which has received much concern because of their unique defense system, namely glucosinolate-myrosinase system. In plant, myrosinases and their substrate glucosinolates are stored in different cell types, myrosin cells and S-cells, respectively, or inseparate intracellular compartments.Myrosinases and glucosinolates are brought into contact upon mechanical damage, herbivore attack or pathogenic fungal and bacterial infections, resulting in the production of a variety of toxic degradation compounds including isothiocyanates, thiocyanates, nitriles, epithionitriles, and oxazolidine-2-thiones. These degradation products have specific functions in many aspects, such as plant defence and nutrition, plant growth and cancer preventive in human.Six myrosinase genes have been reported in Arabidopsis thaliana, including TGG1, TGG2, TGG3, TGG4, TGG5, and TGG6. The TGG4 and TGG5 genes were exclusively transcribed in root tissues while the TGG1 and TGG2 genes were transcribed in leaf, cotyledon, stem, and flowers. TGG3 was a pseudogene, and was specifically expressed in stamen and petal. Similar to TGG3, TGG6 gene contained several frameshift mutations and could not encode an intact peptide, and was a flower specific pseudogene.Myrosinase gene TGG1 of Arabidopsis thaliana was cloned and overexpressed in Pichia pastoris. The recombinant protein of TGG1 was purified with Ni-NTA.The recombinant protein has a molecular weight of 78 kD, larger than the deduced naked protein (58 kD), but similar to the molecular weight of the natural TGG1 protein. The TGG1 signal peptide had some secretion function in yeast, resulted in approximately 25% myrosases in the culture medium. The TGG1 recombinant protein was active in a wide pH and temperature ranges. The optimal reaction temperature was around 40℃, and the pH between 6-9. Myrosinase activity was activated by low concentrations of ascorbic acid, and suppressed by high concentrations. The apparent Km and Vmax were 65μmol/L and 3.28μmol·min-1·mg-1, respectively when sinigrin was the substrate.