Cloning And Sequence Analysis Of Genes Encoding Laccase, The Key Enzyme, In Sclerotium Forming Of Inonotus Obliquus

Inonotus obliquus is quite a valuable medicinal fungus, of which the main medicinal part is sclerotium. In this paper, Laccase coding sequences of11strains including9strains I. obliquus and2strains in the same genus were cloned, which as preliminary study, to explore the molecular mechanism of sclerotium formation on a molecular level and more to provide theory basis for industrial cultivation. In the meanwhile, the cDNA fragments of strain FL were also cloned, which helped to confirm the intron sites and deduce amino acids, and bioinformatics analysis were also conducted on those sequences. Results are shown blow.1. High quality genome DNA of11strains were extracted by improved CTAB method, which then were taken as template, using degenerate primers Cu1F and Cu2R designed based on conserved structural domain of Laccase, to amplify the Laccase coding sequences by PCR reactions. And a totally of18coding sequences around250bp from11strains were cloned.2. Strain FL were induced by3,5-dihydroxytoluene, o-phenylenediamine and benzidine in submerged culture, of which the total RNA were extracted by TRIzol method and then reverse transcribed to synthesized to cDNA. Taken cDNA as template, with using primers CulF and Cu2R, two cDNA fragments of142bp and148bp were amplified by PCR in those induced by3,5-dihydroxytoluene and o-phenylenediamine.3. All the DNA and cDNA fragments were sequenced and aligned, which indicate that the DNA sequences of each strains contain two introns, whose locations are conserves. The Laccase coding sequences with high similarities were sorted in two groups named as Lac1and Lac2, those with relatively low similarities were named as "strain NO." plus "Lac" or "Lac_Un" respectively, and all the sequences are belong to the same gene family. In addition, several sequences of strains in group Lac1or Lac2are completely the same.4. Amino acids were deduced base on the DNA sequences without introns, and all the sequences could encode47amino acids except NBRC9788for46. Conserve sequences were shown in the both ends in which three histidine(H) binding with copper ions were detected, when aligning the amino acids sequences. Amino acids corresponding to Lac1or Lac2are highly agreed within group, showing conservative properties of this gene on expressing level.5. Phylogenetic trees were constructed base on DNA sequences and deduced amino acids sequences respectively by using Neighbor-Joining method. And the results suggest that strain ELS possess a relatively farther genetic relationship with other strains, also considering the presence or absence of different genes of the same gene family, referring to which the strain ELS is absent of both Lac1and Lac2, which indicate that ELS strain remains a higher evolutionary level in I. obliquus. All of these, demonstrate the potential of Laccase coding sequences applying in phyletic classification as assistant molecular marker.6. The cDNA ends of FL strain Lac1gene were amplified through RACE, and two bands of5’ and3’ ends which correspond to expected sizes were detected.5’ sequence were sequenced, and a termination codon of UGA were discovered at the28th base, of which the reason is still unknown.