Cloning,Expression,Purification And Enzymatic Characterization Of AtSK,AtFMN And AtFHY

Herbicides play an indispensable role in the development of modern agriculture.In recent years,the environmental problems have become increasingly serious due to the extensive usage of herbicides.At the same time,the species and quantities of herbicide-resistant weeds have increased rapidly because of the limited number of targets.Therefore,it is necessary to discover novel herbicides targets,which is helpful for new herbicide design.Herbicide targets are key enzymes of important physiological metabolism of plants.Kinase participates in important physiological activities,such as cell growth and metabolism of plants,which provides possibilities for the discovery of novel herbicide targets.In this thesis,three kinase proteins(AtSK,AtFMN,AtFHY)from Arabidopsis thaliana were selected as targets,9 recombinant plasmids were successfully constructed by the protocol of gene clone.Their expression conditions were optimized and screened.Some inclusion body proteins were renatured through refolding after dissolving with the denaturation solution,these proteins were isolated and purified by Ni-NTA column.The catalytic activities of proteins were measured by reverse-HPLC.It was confirmed that,the AtLSK,which was reported as glucokinase(AtGK)previously,has the same catalytic activity as shikimate kinase(AtSK1).The recombinant AtFMN and AtFHY has riboflavin kinase activity and flavin mononucleotide hydrolase activity,respectively.Meanwhile,kinetic parameters and pH,temperature optima and metal ion selectivity for these three proteins(pET-28a-AtLSK,pET-28a-AtFMN,pBG102-AtFHY)were determined.The results were found that,the AtLSK has showed the highest activity at 55℃,pH 7.5,in the presence of Ni2+.The apparent affinity of AtLSK for substrate ATP and shikimic acid(Km value)were calculated as 0.31 mmol/L and 0.38 mmol/L,respectively.The AtFMN has showed the highest activity in the presence of Co2+,the optimum pH and temperature for the AtFMN were 9.0 and 45℃.The apparent kinetic parameters(Km value)for substrates ATP and VB2 were calculated as 0.16 mmol/L and 0.19 mmol/L,respectively.Similarly,the AtFHY hydrolyze the FMN most effectively at 45℃ and at pH 6.0,in the presence of Mg2+.The Km value calculated for the substrate FMN was 0.57 mmol/L.