Cloning Of A Laccase Gene From White-rot Fungi D1 And Constructing The Expression Plasmid

Laccases is a kind of Cu-contained polyphenol oxidase produced by many white-rot fungi. It can degrade 1ignin and phenoxy containing herbicide and waste water in petroleum industry andaffect toxic phenolic materials, so laccases can be widely applied in paper making industry,drinking processing and environment protection. Most of white-rot fungi propagate slowly in water and agglomerate quickly, so they can only live on the surface of water, these characteristic directly restrict their applied for waste water treatment. Research on the cloning laccase structure gene of white-rot fungi has an important significance for cultivate engineering bacilli, which express high laccase activity and treat waste water containing phenolic materials by those engineering bacilli. In this paper, the activity white-rot fungi laccase of laccase in white-rot fungi D1 was mensurated, the sequence of gene have been cloned and analyzed. Main results as followed:(1) The activity of laccase from white-rot fungi D1 was mensurated. The results indicated that it have the activity of laccase, And activity strong.(2) The rapid method of RNA extraction from mycelial fungi were established. Entire and high purity RNA can be isolated by this method.(3) The full-length cDNA of white-rot fungi D1 laccase gene was cloned. The full-length cDNA of white-rot fungi D1 laccase gene was isolated by using RACE (rapid amplification of cDNA ends) technique and Genome Walking. The full length of sequence was 1910 bp, containing an open reading frame(1557 bp), encoding a peptide of 518 amino acids. 5'untranslated region was 51 bp, 3'untranslated region was 302 bp. BLAST analysis revealed that the laccase cDNA and amino acids sequence from Pycnoporus sanguineus shared high identity with laccase gene from other organisms in nucleotides and amino acids respectively.(4) The complete DNA sequence of laccase from white-rot fungi D1 was obtained. the complete DNA sequence of 2124 bp, Besides the coding area of cDNA, the sequence containing 10 introns regions, with length between 52-70 bp, all the...