Molecular Cloning And Differential Gene Expression Of The Global Regulator LaeA In Ganoderic Acid Biosynthesis Process

Ganoderma is one kind of traditional medicine in China, modern medical research found that Ganoderic acid (GA) has anti-tumor, anti-HIV and other important biological functions. Parts of biosynthetic pathway of GA have been revealed, and the relationship between the accumulation of GA and spores formation in Ganoderma lucidum were closely related. The LaeA usually regulates the production of secondary metabolite and the expressions of multiple secondary metabolism gene clusters in filamentous fungi. So the biosynthesis of GA may also be affected by the global regulator LaeA. However, the LaeA gene of G. lucidum has never been reported until now.In this study, the complete expressed sequence tag (EST) database of G. lucidum was used for’in silico cloning’. The known LaeA protein sequence of Penicillium citrinum was chosed as a probe, via the Basic Local Alignment Search Tool (BLAST), sequence assembly, sequence verification, sequence extension and other in silico cloning methods to obtain the cDNA sequence of G. lucidum LaeA. The full-length of cDNA was 1137 bp which was verified by PCR and sequencing. They had high homology with LaeA genes in other speices. The whole gene of LaeA also was found from Ganoderma genome database based on the cDNA sequence. The LaeA gene contains seven exons and six introns. The eukaryotic gene promoter elements including TATA box, CCAAT enhancer binding region and GC box were analyzed. The basic physical and chemical properties, hydrophobic/hydrophilic, subcellular localization, transmembrane domain structure, signal peptide, secondary structure, tertiary structure and evolutionary relationships of LaeA protein were analyzed by various bioinformatics methods, computer and related bioinformatics softwares. The cDNA of LaeA encoded 378 amino acids, which have a relative molecular mass of 42895.3 Da and an isoelectric point value of 7.45. The LaeA is a hydrophilic protein and locates in the cytoplasm. It has S-adenosine methionine (SAM) binding sites, and contains a typical super family structure domain of methyltransferase using SAM as the substrate, so it would be have the function of methyltransferase. It was highly conservative among LaeA from several different white-rot fungi species, especially had high homology with Coriolus versicolor and Dichomitus squalens.In order to further study the properties of the LaeA protein in G.lucidum, the LaeA gene was linked with PET32a plasmid and then transfered to E. Coli BL21 (DE3). The heterologous gene expression system of LaeA in E. coli was constructed. The LaeA protein was expressed in E. Coli after being induced by IPTG. It will afford a good foundation for the further study of protein structure and function of LaeA.Submerged cultivation and a two-stage liquid static cultivation were main cultivation modes for the production of GA by G. lucidum. The production yield of GA and the relative expression level of LaeA during the two processes were detected by HPLC and real time PCR. Data showed that the content of GA in cell from the liquid static culture was obvious higher than from submerged culture. The correlation between transcription expression level of LaeA gene and the yield of GA showed significantly positive.The discovery of global regulatory factor LaeA in G. lucidum will provide a clue for understanding the molecular regulatory mechanism in GA biosynthesis.