Cloning,Expression And Enzyme Activity Analysis Of Chorismate Mutase(OHCmu)Gene Of Oidium Heveae

powdery mildew in rubber tree,caused by Oidium heveae B A.Steinmann,is one of the most important leaf diseases,greatly reduced rubber production while serious harm.Oidium heveae belongs to obligate parasitism fungus,which can not be cultured in vitro.The transformation technology of Oidium heveae is under developing,so the pathogenic mechanism of Oidium heveae is still less.Chorismic acid is the final product of the Shikimic acid metabolism pathway and widely exit in plants,bacteria,fungi and animals.Chorismic acid is involved in the synthesis of aromatic amino acids(Phe、Tyr、Trp)and is related to the synthesis of plant defense-related hormones(SA、IAA etc),which play a very important role in plant disease resistance.During infection,plant pathogens can secrete chorismate mutase into plant cytoplasm and affect plant shikimate pathway,thereby inhibiting plant defense and promoting the colonization of pathogens.In this study,A Chorismate mutase gene OHCmu of the Oidium heveae was cloned and identified,and GST-OHCmu fusion expression vectors was constructed.OHCmu enzyme activity was determined and properties were analyzed.This study will lays the foundation for further study on the function of OHCmu in the interaction between pathogens and hosts and for understanding the the pathogenic mechanism of Oidium heveae.The main results of this study are as follows:1.Two different Chorismate mutase genes were obtained from NCBI and PDB databases.A Chorismate mutase gene sequence(named as OHCmu)was gotten in the genome of Oidium heveae in our laboratory by homology comparison.Then it was amplied by PCR and RT-PCR.The results of the sequence analysis showed that the gene size of OHCmu was 843 bp contained 1 intron and encoded 262 amino acids.It has d5csma_domain and belongs to Chorismate mutase Ⅱ family.The results of quantitative real-time PCR showed that the expression of OHCmu gene increased in the process of Oidium heveae infecting rubber tree.The expression reached the highest level at 13h,and then decreased gradually.2、The prokaryotic expression vector of GST-OHCmu fusion gene was constructed.The inducing conditions were optimized,the GST-OHCmu fusion protein was well expressed by 0.8 mmol/L IPTG at 16℃.And the size of the fusion protein was approximately 56 KD.The purity OHCmu protein was obtained after purification by GST affinity chromatography and the GST tag was cleaved.3、The activity of OHCmu was tested by protease activity assay,and the effects of temperature and product(trp,tyr,phe)on enzyme activity were analyzed.The results showed that the purified OHCmu has enzymatic activity,and its enzyme activity is not affected by trp,tyr and phe.But its activity was affected by temperature,and its activity almost lost at 45℃.4、Key enzyme active sites of OHCmu were confirmed by point mutation.The results showed that the 163rd arg and 173rd lysine were the key enzyme active sites of OHCmu,which were consistent with the reported Cmu enzyme active sites of Ustilago maydis,indicating that the enzyme active sites were relatively conservative.