Bioinformatics Analysis And Functional Validation Of Laccase Genes In Magnaporthe Grisea

Laccases (EC 1.10.3.1) are multi coppper-containing phenol oxidases and be able to catalyse the reaction of oxygen of electroreduction phenolic materials. Laccases have been assigned several different biological roles. In higher plants, laccases are involved in lignification of xylem tissues. In fungi, laccases are linked to pigment biosynthesis, fruitbody morphogenesis, lignin degradations condial pathogenicity and detoxification. It can degrade lignin and phenoxy containing herbicide and waste water in petroleum industry and affect toxic phenolic materials, so laccases can be widely applied in paper making industry, drinking processing and environment protection. The function of laccases is still undetermined in the rice blast fungus. The thesis based on the bioinformatics studying of the laccase gene in Magnaporthe grise, cloned one of the genes and over-expressed this gene in M. grisea.In NCBI GeneBank we have gotten the conserved amino acid sequence of the laccase of the Neurospora crassa. BlastP the sequence with the genome sequence of the Magnaporthe grise, 13 related genes were found. Among them only MG09139.4 has no intron. In comparison with the EST library, MG09139.4 and MG07771.4 hit to EST library of M. grisea. SignalP3.0 prediction results showed that MG07771.4 almost has no the probability to be a Signal peptide probability, the probability of that MG09139.4 was a signal peptide is 99.5%, of which the cleavage site is most likely between pos. 18 and 19. TMHMM results indicated that the expected number of amino acids intransmembrane helice is 2.83118, and the possibility of N terminus on the cytoplasm membrane is 0.13339. ProtComp v6.0 results showed that MG09139.4 was an Extracellular (Secreted) protein. PredictProtein results showed protein secondary structures: a-helix 10.385%, P-sheet 17.586%. Predicting the conserved domains of the laccase, there are 116 conserved amino acids, and the homology to one Multicopper oxidase (gnl|CDD|25500) in Pfam is up to 96.8%, and 88.9% to another Multicopper oxidase (gnl|CDD|11840) . DNAstar results indicated that: the protein comprised of 597 amino acids, including 198 hydrophobic amino acids, and 191 polarity amino acids. The molecular mass of the protein is 65980.78Da with 6.66 Isoelectric Point .The amino acid sequence of laccases in GenBank were aligned and to construct a phylogenetic tree.The position of M.grisea laccase proteins in the evolution were successfully predicted in this tree.The Laczcz gene which encodes MG09139.4 protein was cloned from M. grise strain 70-15. The over-expression vector pTE11-LAC was constructed by inserting the Laczcz gene at the XhoI and SpeI sites of the eukaryotic expressing vector pTE11 under the control of RP27 promoter. The PEG mediated transformation method was used to transform the vector to M.grisea, and 2 transformants were gotten .These can help us to find the function of laccase in M.grisea.