Expression Pattern Analysis And Enzymatic Analysis Of Trehalose Metabolizing Enzyme Genes In Magnaporthe Oryzae

The trehalose metabolizing enzyme gene of Magnaporthe oryzae is closely related to the pathogenicity of Magnaporthe oryzae.The study of trehalose metabolizing enzyme gene and its enzyme activity characteristics can provide a theoretical basis for the control of rice blast fungus.In this experiment,the functions of trehalose metabolizing enzymes were analyzed from the aspects of Magnaporthe oryzae stress,prokaryotic inducible expression,enzymatic properties and subcellular localization.The main research results were as follows:1.qRT-PCR was used to detect the expression patterns of trehalose metabolizing enzyme genes of M.oryzae under high temperature and low temperature stress conditions.The results showed that under high temperature stress,the expression levels of trehalose synthase and trehalase did not change significantly at the early stage of stress,but were significantly expressed at the later stage of stress.2.In order to study the enzymatic properties of trehalose-6-phosphate synthase（MGG＿03860,Tps1）,MGG＿03860 was successfully cloned,and the prokaryotic expression vector p ET-30a-MGG＿03860 was constructed.After induction for 16 h,a58 k Da soluble target protein was obtained.The target protein was purified and obtained,and the enzyme activity of the target protein was detected by trehalose synthase reaction,which was 33.2 U/mg.At the same time,the optimum temperature for the enzymatic reaction of Tps1 was 30℃,and the optimum p H was 7.When exploring the effects of different ions Mn²⁺、Co²⁺、Zn²⁺、Ca²⁺、K⁺、Mg²⁺、Ni²⁺and Cu²⁺at the same concentration of 10 m M on the activity of Tps1 fusion protease,it was found that Mn²⁺、Zn²⁺and Cu²⁺had a certain inhibitory effect on Tps1 enzyme activity.It was found that Co²⁺、Ca²⁺、K⁺、Mg²⁺and Ni²⁺had a certain promoting effect on Tps1 enzyme activity,among which Mg²⁺had the most significant promoting effect on Tps1 enzyme activity,indicating that Mg²⁺was the most suitable cation to activate Tps1 enzyme activity.And the Km constant of recombinant Tps1 enzyme to G-6-P is（K_m）1.4120 m M and V_max is 41.3223 m M/min,the K_m constant（K_m）of recombinant Tps1 enzyme to UDPG is 15.2439 m M and V_max is 61.3497 m M/min.At the same time,in order to study the subcellular localization of Tps1 protein,the p BT3-SUC-MGG＿03860-GFP vector was constructed in this experiment and transferred into NMY51.All these laid the foundation for the follow-up study of the function of Tps1 protein.3.In this study,two trehalase genes were successfully cloned:MGG＿01261（TREl）and MGG＿09471（NTH1）.The prokaryotic expression vectors p ET-30a-MGG＿01261 and p ET-30a-MGG＿09471 were also constructed.The target protein could be obtained after 16 h of induction under conditions,among which the MGG＿01261 protein was less expressed in the supernatant,with a size of about 78KDa.The MGG＿09471 protein is an inclusion body protein with a size of 85 KDa,but it is difficult to obtain in the supernatant,so it cannot be used for subsequent purification,and the conditions need to be further changed in order to obtain a soluble protein.Then,the enzymatic properties of soluble protein were analyzed and studied,and the optimum temperature of recombinant protein was 50℃and the optimum p H was 6.When exploring the effect of different ions Mn²⁺,Co²⁺、Zn²⁺、Ca²⁺、K⁺、Mg²⁺、Ni²⁺and Cu²⁺on the activity of recombinant protease at the same concentration of 10m M and 5 m M,it was found that manganese ion had the strongest promotion effect on enzyme activity,no matter at low concentration In the case of high concentration,Cu²⁺、Ni²⁺and Mg²⁺all inhibited the enzyme activity.The results showed that Mn²⁺was the activating ion of trehalase MGG＿01261,which laid a foundation for the subsequent study of the function of MGG＿01261 protein.And the Michaelis constant K_m of trehalase was 1.8 m M and V_max was 1.66 m M/min.