Cloning, Expression Of Myrosinase Gene TGG5 In Arabidopsis Thaliana And Research Of Myrosinase Activity

Myrosinase widely exists in the Cruciferae plant, which can catalyse the hydrolysis of thioglucosides. A unique defense system is composed of myrosinase and glucosinolate, namely glucosinolate-myrosinase system. myrosinase and glucosinolate are mixed upon infestation by pests, and thioglucosides are hydrolyzed into toxic compounds to serve as defense weapons, called "mustard oil bomb (mustard oil bomb) ". In addition to play an important role in biological defense, but also the system has a special role in food flavor, human nutrition and cancer Prevention.Now, six myrosinase genes have been found in Arabidopsis, which is respectively TGG1, TGG2, TGG3, TGG4, TGG5 and TGG6. Myrosinase gene expression shows a specific time and space. The TGG1 and TGG2 genes were transcribed in cotyledons, stems, leaves, flowers and silique, but not in roots. TGG3 is a pseudogene, only expression in flower. TGG4 and TGG5 only were specifically transcribed in the roots of Arabidopsis. TGG6 just like TGG3, is also a pseudogene. In this paper, RT-RCR detection confirmed the TGG5 gene only specificlly expressed in roots, but not in cotyledons, stems, leaves, flowers and silique, This result explains the activity of the root in Arabidopsis.In this research, the cDNA sequence of TGG5 was cloned, and the pichia expression vector pPIC3.5K was constructed, then transformation of pichia GS115 was made. The expression of recombinant TGG5 protein was induced by Methanol, and purified by Ni-chelating colum. SDS-PAGE analysis showed that the recombinant protein concentrated in the 58KD molecular weight around and showed typical features of protein glycosylation with a more visible tail. The myrosinase activity of TGG5 recombinant protein was future researched after separation and purification. The results indicated that the Km and Vmax of the enzyme were 1.96 mmol/L and 0.084 mmol·min-1·mg-1 respectively. The optimal PH of tested myrosinase was 5.5. The myrosinase showed the wider adaptability in the temperature range of 37-80℃, and took on better stability on 80℃high temperature. NaCl has an obvious inhibitory effect on activity of tested myrosinase, but low concentration of ascorbic acid can activate the myrosinase, which reached to a climax with an ascorbic acid concentration of 1.5 mmol/L.