Overexpression Of Key Enzyme Genes In Biosynthesis Pathway Of Ganoderma Lucidum Polysaccharide

Ganoderma lucidum is a famous fungus with high healthy and medicinal functions.As the main active ingredients of G.lucidum,polysaccharides have numerous biological activities including regulating immunity,anti-tumor,anti-oxidation,anti-HIV and lowering blood pressure.Polysaccharides are mainly produced from the fermentation broth,mycelia and fruit body.At present,most researches on polysaccharides in G.lucidum focus on increasing yields through fermentation control technology,while the concern about regulating metabolism of polysaccharide synthesis is lacked through molecular level.In this study,the recombinant plasmids were constructed and the key enzymes,phosphoglucomutase(PGM)and phosphoannose isomerase(PMI),in the metabolic pathway of polysaccharides were overexpressed by Agrobacterium tumefaciens(ATMT).The expression of the key enzymes gene at the transcriptional levels was detected by qRT-PCR.The yields,monosaccharide compositions and related enzymes in synthetic pathway of G.lucidum polysaccharides were also studied.In addition,the differences between the wild type and recombinant G.lucidum strains in cell morphology and cell ultrastructure were observed by scanning electron microscope(SEM)and Transmission Electron Microscope(TEM)techniques.The main results are as follows:1.The gene sequences of PGM and PMI in G.lucidum strain were found in NCBI.The primers were designed by DNAMAN.The total gene of G.lucidum was used as template to amplify the target gene fragments by PCR.The recombinant plasmids,PJW-PGM and PJW-PMI,were constructed by homologous recombination based on the pCAMBIA1301,and were transformed to protoplasts of G.lucidum by ATMT.The recombinant strains was screened from the plate containing the corresponding antibiotic.2.With the wild-type G.lucidum strain as control,the results showed that the bacterial biomass of G.lucidum recombinant strains were improved to some extent compared with wild type,and consumption rate of reducing sugar was faster.The results about the composition and proportion variance of polysaccharides in the fermentation process showed that due to the overexpression of pgm and pmi genes,the glucose flowed to different synthetic metabolic branches and affected the monosaccharide compositions and proportions of polysaccharides in G.lucidum.The key enzymes involved in the synthesis of sugar nucleotide donors were verification by RT-PCR at the transcription level.The RT-PCR results showed that the overexpression of pgm and pmi genes not only made the relative expression at mRNA level higher than that of wild-type strain,but also promoted the upstream and downstream gene expression.With the wild-type strain as control,the change of specific activity of the enzymes involved in the synthesis of sugar nucleotide donors in the polysaccharides synthetic pathway was also studied.The results showed that the specific activity of PGM and PMI in the recombinant strains was higher than that in the wild type strain,andt also promote the activity of PGI,UGPG of polysaccharide synthesis pathway.3.With the wild-type G.lucidum strains as control,the recombinant G.lucidum strains were analyzed by SEM and TEM technique to observe the differences in their cell morphology and ultrastructure.The results showed that in SEM the polysaccharides covered on the cell surface in recombinant strains are more than those in the wild-type strains while the differences in the cell ultrastructure are not significant in TEM.