| Ganoderma lucidum is one of the traditional Chinese medicine,which has a long history of medicinal use.G.lucidum polysaccharides are macromolecular substances extracted and separated from fruiting body or liquid submerged fermentation.It is one of the main bioac tive substances of G.lucidum.Because of its important pharmacological effects,the regulation mechanism of G.lucidum polysaccharides synthesis has become a hot spot in recent years.Alternative oxidase(AOX)is the main oxidase of mitochondrial alternat ive respiration pathway,which affects the production of reactive oxygen species in the electron transport pathway.Reactive oxygen species(ROS)are by-products of the metabolic process of organisms,which can be used as signal molecules to react with redox proteins to regulate the transcriptional expression of related genes in cells.In this paper,the engineering strain of G.lucidum aox gene silencing and overexpression was constructed to explore its regulatory role in polysaccharide synthesis and its effect on the structure and activity of polysaccharides.In this study,the gene aox of alternative oxidase related to reactive oxygen species was cloned in G.lucidum,and the engineering strains of aox gene silencing and overexpression were constructed res pectively.The fluorescence quantitative results showed that the aox gene expression of overexpressed strains OE-AOX-2 and OE-AOX-21 was 2.02 and 1.75 times higher than that of the control group,while the silencing efficiency of aox gene of silencing stra ins AOXi3 and AOXi25 was 68% and 86%,respectively.Through liquid shake flask fermentation,the polysaccharide yield of silent strain increased significantly,while that of overexpressed strain decreased significantly.Furthermore,the expression levels of enzyme genes(pmi,pgm)related to polysaccharide synthesis were detected,which was consistent with the trend of fermentation results.The expanded culture of 5 L fermentor was carrie d out under the same conditions and compared with the wild type,it wa s found that the polysaccharide yield of AOXi25 strain was 3.46 g/L,which increased by53.7%,while OE-AOX-21 strain was 1.82 g / L,which decreased by 20%.The mycelia of G.lucidum were incubated with DCFH-DA probe.The results showed that the intracel lular reactive oxygen species levels of AOXi3 and AOXi25 strains were 1.78,1.96 times higher than the wild type,while those of overexpressed strains OE-AOX-2 and OE-AOX-21 were 0.63,0.44 times,respectively.Furthermore,the activity of antioxidant syst em-related enzymes SOD,CAT,APX and GSH content,and the changes in m RNA transcription level of genes related to active oxygen scavenging system were determined.It was found that the activity and transcriptional expression level of antioxidant enzymes increased to different degrees in silencing engineering strains,but on the contrary in overexpression engineering strains.It was found that there was a significant positive correlation between the polysaccharide yield and ROS level of silenced and overexpr essed engineering strains,indicating that aox gene can regulate the synthesis of G.lucidum polysaccharides by regulating the level of intracellular ROS.Extracellular polysaccharides from different engineering strains were collected and purified to obtai n six polysaccharide components: WE-1,WE-2,i E-1,i E-2,OE-1 and OE-2.The primary structure of polysaccharides was analyzed by infrared spectroscopy and gas chromatography-mass spectrometry.The scavenging rates of polysaccharides on DPPH,ABTS and hydro xyl radicals were tested.the results showed that the scavenging intensity of acidic polysaccharides was higher than that of neutral polysaccharides in vitro,and all of them had different scavenging effects on free radicals,indicating that they all had certain antioxidant activity.Among them,the scavenging effect of acidic polysaccharide i E-2 of silent strain on hydroxyl radical was the best,and the scavenging rate reached 52.08% when the concentration was 2.5 mg/m L.In this study,it was found that AO X participated in the regulation of reactive oxygen species on the biosynthesis of G.lucidum polysaccharides.By regulating the level of intracellular reactive oxygen species,the expression of genes related to the biosynthesis of G.lucidum polysaccharides was regulated,and then the biosynthesis of polysaccharides was regulated.To study the regulatory effect of aox gene on the biosynthesis of G.lucidum polysaccharides,it is helpful to better understand the regulatory mechanism of fungal secondary metabolism. |
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