Breeding And Gene Expression Of Glucose Metabolism Of Lepista Sordida With High Exopolysaccharide

Lepista sordida is a kind of grass rot fungus,that contains essential trace elements,low fat,high protein make it have great edible value,in addition,because of polysaccharides for Lepista sordida has antioxidant,anti-tumor and anti-aging effects,which have great medicinal value.However,at present,in China,the wild resources of the Lepista sordida are scarce,the excellent germplasm resources that can be cultivated are scarce and the artificial cultivation is difficult,resulting in its large-scale cultivation and industrialization development and utilization are seriously restricted.On the one hand,the selection and breeding of high-yield polysaccharide Lepista sordida strains not only enrich the edible mushroom germplasm resources,but also promote the development and utilization of the Lepista sordida and its polysaccharides.On the other hand,there are very few reports at home and abroad on the screening of reference genes for Lepista sordida and the study of the metabolic mechanism of polysaccharides from the perspective of gene expression.To this end,the following studies have been carried out:(1)Taking the LS01 strain of Lepista sordida as the experimental object,the protoplast monokaryonoid strain LS01?10 was prepared,and then the LS01?10-based strain was used as the starting strain,and the high exopolysaccharide yield of the Lepista sordida strains were selected by plasma mutagenesis.Under the condition of mutagenic power of200 W and mutagenic time of 60 S,50 mutagenic strains were preliminarily obtained,and 5high exopolysaccharide yield mutagenic strains with stable traits were screened,and the content of extracellular polysaccharides was 1.2-2.4 times that of the starting strain.The hyphae morphology and color of the mutagenic strains have changed to some extent,and the biomass is higher than that of the starting strain.ISSR experiments have shown that mutagenic strains undergo varying degrees of genetic changes compared to the starting strain.The mutagenic strains with high exopolysaccharide yield provide an important germplasm resource for the preliminary analysis of the mechanism of high exopolysaccharide yield.(2)By measuring the ability of extracellular crude polysaccharides to scavenge DPPH free radicals,hydroxyl radicals(·OH)and ABTS cationic free radicals,the in vitro antioxidant activity of Lepista sordida was evaluated and analyzed.The results show that under the same sample concentration,with glucose,sucrose,mannitol and maltose as carbon sources,there are differences in the antioxidative activity in vitro of different Lepista sordida strains,when taking mannitol as carbon source,LS01?10?B reaches 91.7% to ·OH scavenging rate at the highest When taking glucose as carbon source,LS01?10?B reaches25.0% to DPPH scavenging rate at the highest;but has no scavenging effect to ABTS cation.Glucose can be used as the best carbon source for the crude polysaccharide to scavenge DPPH free radicals in the mutant strain of Lepista sordida,and mannitol can be used as the best carbon source for the crude polysaccharide to scavenge hydroxyl radicals(·OH)in the mutant strain of Lepista sordida.(3)Using fluorescence quantitative PCR technology to screen the most suitable internal reference of Lepista sordida from 9 candidate internal reference genes,and detects11 key genes of polysaccharide synthesis pathway in Lepista sordida high-yielding exopolysaccharide mutant strain and its starting strain and 7 polysaccharide metabolic pathway gene expression levels.The results showed that the ribosomal protein L4(rpl4)and glyceraldehyde-3-phosphate dehydrogenase(gapdh)had high expression abundance and good amplification efficiency,and thus were selected as ideal internal reference genes for Lepista sordida.Compared with LS01?10?9,five key genes(GK,GMD-3,UGE,UGDG and PFK)involved in polysaccharide synthesis and four genes(GT-1,GT-2,NOX-3 and NOX-4)involved in polysaccharide metabolism were significantly up-regulated in LS01?10?9.The mechanism of exopolysaccharide production in mutated plants was discussed from the perspective of gene expression at genetic level.