Bioinformatics Analysis And Cu²⁺ Inducing Expression Of Putative Laccase Genes From Ganoderma Lucidum Genome

Ganoderma lucidum is a basidiomycete white rot fungus, to date, little is known about the enzymes in lignin degradation and utilization. With the publication with genome data of G. lucidum, it is available to note and analysis the lignin-cellulose degradation enzyme coding genes in fungi at genome level. However, the gene annotation at genome scale only can explore the proteins which have homology with the present amino acid sequences of lignin-cellulose degradation enzymes, the novel lignin-cellulose degradation enzymes have to be depended on the function experiments. Hence, this study analyzed the putative laccase genes Lacc1-Lacc16and their deduced amino acid sequences by using bioinformatics methods, Cu2+as inducer to induce laccase isoenzymes synthesis and secretion, the expression of Lacc1-Lacc16were quantitate and analyzed by Real-Time quantitative Polymerase Chain Reaction(RT-qPCR). We intend to unveil the correlation between promoter sequence structure and transcriptional regulation in G. lucidum laccase gene, additionally, provide a foundation for exploring the regulation mechanism of laccase genes and the subsequent research on laccase genes functional genomics. The results are as followed:1. SignalP result shows that Lacc2, Lacc7and Lacc11have no signal peptide. The Blastp result uncovered that amino acid sequences of Lacc1-Lacc16were contain3conserved Cu-oxidase domains. It worthy noting that Lacc12had highest identity with Fet3protein from Dichomitus squalens LYAD-421SSI, and the Multiple sequence alignment result display that amino acid sequence of Lacc12lost a conserved cysteine in the laccase signature sequence, moreover, Lacc12had long distance with other15putative laccases amino sequence, above all, Lacc12is very likely a ferroxidase coding gene.2. The expression of laccase isoenzymes was induced during the G. lucidum growth with adding final concentration150μmol/L Cu2+into medium, the laccase activity was upregulated1.52,2.86,2.07and4.00fold at4th,6th,8th and12th day, respectively.3. Bioinformatics analysis revealed that the promoter regions of Lacc1-Lacc16include various cis-acting elements, such as metal responsive element(MRE), stress responsive element (STRE), CreA-binding site, nitrogen binding site(NIT), xenobiotic responsive element(XRE), antioxidant responsive element(ARE), and core promoter elements TATA-box, CAAT-box. The expression analysis of putative laccase genes shows that Laccl、Lacc2、Lacc3、Lacc6、Lacc9gene were strongly upregulated by Cu2+, meanwhile their promoter regions were contain MRE, suggested that metal responsive induction pathway were existed in this genes. However, Lacc4and Lacc10barely induced by Cu2+at different developmental stage of G lucidum, although their promoter regions contain several MRE, therefore, a deduction that the number of MRE in promoter region have no direct correlation with the inducing effect on laccase gene at transcriptional level.4. Laccl、Lacc2、Lacc9、Lacc11、Lacc14、Lacc15genes were upregulated by Cu2+at specific growth phase of G lucidum, we speculated two possible reasons for this phenomena, one is that the variation in medium composition occurred during growth cause specific expression; another is that these putative laccase genes encoding different proteins play diverse physiological functions in G lucidum.5. Lacc7、Lacc9、Lacc11、Lacc14genes were little expressed in control group and Cu2+induction group, this case could be accounted for these genes was gene redundant copies during evolution, which widespread among fungi and plant laccase gene family.6. The4th day of G. lucidum growth, the expression value of Lacc8gene was the higest among control group and Cu2+induction group, in the meantime, the highest laccase activity of control group reached97.20U/mL at4th day, the highest laccase activity of Cu2+induction group reached165.04U/mL at6th day. Combined the expression profile of Lacc8and laccase expression profile, Lacc8was supposed have close relation with the synthesis of extracellular laccases.