Purifition Of Enoyl-CoA Hydratase And Analysis It’s Interaction With Protease MAP From Magnaporthe Oryzae

Magnaporthe oryzae as one of ascomycete fungi can infect a variety of grasseslike rice, wheat, barley and other, leading to plague. Rice blast disease is a persistentthreat to the global rice harvest, causing substantial crop losses every year. Rice blastalso one of the major diseases of rice in china. Analyze the developmentalmechanisms of infectious hyphae is the important aspects to study the pathogenesis ofM. oryzae. MAP protease is highly conserved among archaea, prokaryotes andeukaryotes, which is is a variety of structures ATP-dependent protease. The MAPprotease was found to play an important role in cellular homeostasis, mitochondrialprotein quality control and metabolic. Enoyl-CoA Hydratase(ECH) catalytic thesecond step of the fatty acid β-oxidation metabolic pathways. Generation of ECH viafatty acid β-oxidation is a prerequisite for appressorium-mediated plant infection. Thepreliminary study found that MAP protease can control the size of the lesion byregulate the infection hyphae extended. On this basis, we obtain a mutant whose haveabnormal expression of MAP protease. We obtain many patener of MAP protease bypurified by6*His-tag pull down. ECH is one of them. The purpose of this research isto purification ECH and identification its interaction with the MAP protease.The main experimental results:(1) Total RNA was extracted from the wild-type strains of M.oryzae in “ZLJ88”.Full length cDNA was obtain form the RNA transcription. The gene MAP and ECHwas cloned from the cDNA. After sequence analysis, it is found that MAP has an openreading frame of3360bp, encoding1120-amino acid residues with a deducedmolecular weight of123.09kD, pI is5.45，and ECH has an open reading frame of873bp, encoding291-amino acid residues with a deduced molecular weight of32.01kD, pI is5.05.(2) ECH was inserted to pET expression vector and induced by IPTG in E. coli.The purification of recombinant ECH from E. coli was performed by Ni-NTA Agarosechromatography method. SDS-PAGE analysis of recombinant ECH revealed a molecular weight of approximately32.01kDa and this protein exists in the form ofinclusion bodies in E.coli. In this form the protein activity is likely to reduce anddisappear. So the switch to the eukaryotic expression vector to study proteininteractions.(3) Construction pPICZB-MAP and pPICZC-ECH vector. The plasmids weretransformed into yeast to construct the eukaryotic expression vector. ECH protein wasextracted through the induction of methanol from yeast which contains recombinantplasmids.(4) The purification of the ECH protein from total proteins of yeast which containsrecombinant plasmids.（5）Yeast two-hybrid analysis interaction ECH with MAP protease.Our research lays a solid theoretical foundation for the study biochemical activityand biological functions of ECH. Yeast two-hybrid analysis interaction ECH withMAP protease. At the same time this work will provide important information forunderstand the molecular mechanisms of rice blast fungus infection hyphae and host-pathogen pathogenic process, and will provide a scientific basis for the effectiveprevention and control of rice blast fungus.