Cloning And QRT-PCR Analysis Of A Laccase Gene From Ganoderma Tsugae

Laccase is a kind of Cu-contained polyphenol oxidase. Laccase has manyfunctions such as lignin degradation, growth promotion, contaminated microbeinhibition and fruiting-body formation, which directly affect the development andmorphology of the occurrence of fungi. The qPCR system for qualitative andquantitative detection of a laccase gene from Ganoderma tsugae has been establishedsuccessfully, which will lay the foundation for the exploring the regulatorymechanism of laccase expression, the relationship between laccase expression leveland biological function, and the feasible qualitative and quantitative detection systemfor monitoring expression levels of other functional genes of Ganoderma tsugae.In this study the degenerate PCR technology and thermal asymmetric interlacedPCR method were employed to determine the genomic DNA sequence of the laccasegene from Ganoderma tsugae, and PCR degenerated primers were designed and usedfor qualitative PCR according to the genomic DNA sequence involved in cDNA ofthe laccase gene. The transcription levels of four candidate internal reference genes indifferent nutrient liquid media were analyzed to screen out the suitable internalreference genes for qRT-PCR. Using qRT-PCR and laccase activity commomdetermination method, the laccase activity and transcription levels were detected inbasic and four modified media to explore the relationship between exocellular laccaseactivity and laccase transcription levels. Main results were as follows:1Cloning of a laccase gene from Ganoderma tsugaeThe sequence1611bp of laccase gene from Ganoderma tsugae was obtainedthrough the degenerate PCR technology. The degenerated primers were designedaccording to the consensus amino acid sequences of fungi laccase gene. The nestedspecial primers on the5'-end and3'-end on the known sequence were designed, andthen a390bp flanking sequence of5'-end and a339bp flanking sequence of3'-endwere obtained by TAIL-PCR. These three sequences were joined together to obtain a 2113bp laccase genomic DNA sequence. Using PCR primers designed according tothe genomic DNA sequence, the cDNA sequence of the laccase gene fromGanoderma tsugae was cloned. The length of cDNA was1566bp. According to thecomparison analysis, nine introns of50-77bp were included and their sequences werein accordance with the rule of "5'-gtnng" and "c/tag-3'", the sequence encoded521amino acids after excluding the introns. Ganoderma tsugae laccase amino acidfragment shares considerable homology of above70%following the analysis withother laccase genes in fungi.2Screening of internal reference genes of qRT-PCR for Ganoderma tsugaeAccording to18S rRNA, β-Tub, RPb2and TEF1cDNA sequences ofGanoderma tsugae, four pairs of primers were designed, and then transcription levelsof four candidate internal reference genes were detected in different nutrient liquidmedia. The results showed that in qRT-PCR analysis, β-Tub, RPb2and18S rRNAcould be employed as internal reference genes to study the laccase transcription levelbased on the analysis with GeNorm, NormFinder and BestKeeper algorithms.3qRT-PCR analysis of laccase transcription levelAccoding to the basic medium, there were four kinds of modified media: basicmedium add Cu2+(0.05mmol/L), basic medium add zhezha (0.1g/100mL), high C lowN medium, and high N low C medium. After9days, laccase activity was detected indifferent media: basic medium8.4U/mL, basic medium add Cu2+23.6U/mL, basicmedium add zhezha55.1U/mL, high C low N medium7.3U/mL, high N low Cmedium23.3U/mL. Laccase showed an transcription pattern: basic medium addzhezha> high C low N medium> high N low C medium> basic medium> basicmedium add Cu2+. The results showed that the exocellular laccase activity was notproportioned to the laccase transcription level, it may be related to enzyme activitywhich cannot be represented all about.