Cloning And Sequence Analysis Of Laccase Gene From White-rot Fungi

Laccases is a kind of Cu-contained polyphenol oxidase produced by many white-rot fungi. 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. 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 of laccase in selected 25 strains were mensurated, the sequence of six white-rot fungi laccase gene have been cloned and analyzed. Main results as followed:(1) The activity of laccase from 25 selected white-rot fungi was mensurated. The results indicated that 11 strains have the activity of laccase, including Phellinus linteus Pycnoporus cinnabarinus Tyromyces zonatulus Daedalea quercina Lenzites tricdor Climacocystis borealis Lenzites betulina Pycnoporus sanguineus Irpex lactea Cerrena unicolor Favolus alveolaris.(2) The rapid methods of RNA and DNA extraction from mycelial fungi were established. Entire and high purity DNA and RNA can be isolated by these methods.(3) The results of RT-PCR experiment showed that mRNA of Eljvngia applanata has no Poly (A) tail sequence.(4) Six specific fragments were obtained from 6 fungi that have laccase activity using a pair of degenerate primer. These fragments share high identity with laccase gene from other organism, which were Pycnoporus cinnabarinus ( 1227 bp ) Pycnoporus sanguineus ( 1227 bp, AY331189) Cerrena unicolor (1207 bp, AY45.4305) Tyromyces zonatulus (1223 bp, AY454306) Climacocystis borealis (1201 bp, AY333124) Daedalea quercina (1232 bp, AY333125) .(5) The full-length cDNA of Pycnoporus sanguineus laccase gene was cloned. According to the laccase gene fragment of Pycnoporus sanguineus (AY331189), The full-length cDNA of Pycnoporus sanguineus laccase gene was isolated by using RACE (rapid amplification of cDNA ends) technique. The full length of sequence was 1902 bp, containing an open reading frame(1554 bp), encoding a peptide of 518 amino acids.5' untranslated region was 51 bp, 3'untranslated region was 297 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. The laccase protein has four catalytic cupric ions domain and seven glycosylation sites, the relative molecular weight was 56313.2, and theoretical pI was 5.59.(6) The complete DNA sequence of laccase from Pycnoporus sanguineus was obtained. Based on the Pycnoporus sanguineus laccase cDNA sequence, a pair of specific primer had been designed; the complete DNA sequence of 2154 bp (AY510604) was isolated by PCR. Besides the coding area of cDNA, the sequence containing 10 introns regions, with length between 52~70 bp, which showed typical eukaryotes intron characteristic, including 5' donor splicing site3' receptor splicing site and bifurcate sequence.