Study Of Carbon Nitrogen Sources And PH On The Polysaccharide Content And Its Biosynthesis Mechanism Of Lentinan In Lentinula Edodes

In recent years,with the improvement of people’s living standards,people are paying more and more attention to their health.Letinous edodes is a medicinal and edible fungus.The lentinan,which is the main metabolites of L.edodes,has many activities such as anti-tumor,immunity enhancement,anti-virus,anti-oxidation,hypoglycemic and so on.It is currently one of the most promising functional food and medicine resources.Therefore,improving the yield of lentinan is of great value.Culture medium is one of the main factors which will affect the production of secondary metabolites in microorganisms.The yield of lentinan is also affected by the composition of medium.Polysaccharide metabolism is a complex biochemical process.To Study its specific mechanism will help to increase the yield of lentinan.Real time fluorescence quantitative PCR(RT-qPCR)is an important method to analyze the gene expression level.However,the reliability of the results depends on many factors,among which suitable internal reference genes will improve the reliability of the results.In this paper,using Xin808,which is a widly cultivated strain,as the experimental materials,we studied the effect of carbon,nitrogen sources and pH on the biomass,intracellular polysaccharides,and extracellular polysaccharides in L.edodes.The stability of 10 housekeeping genes of L.edodes under different environments and conditions was analyzed and then screened the most stable expression internal reference genes.Based on this,we further analyzed the transcript expression levels of three key enzyme genes involved in polysaccharide biosynthesis under different carbon,nitrogen and pH.The relationship between the transcript expression levels of the three key enzyme genes and the polysaccharide content was also analyzed.The main contents and results were as follows:(1)The biomass,intracellular polysaccharide and extracellular polysaccharide content of L.edodes were different under different carbon,nitrogen sources and pH fermentation conditions.Among the six carbon sources,the higest biomass and intracellular polysaccharide were detected when starch was used as the carbon source,and the higest content of extracellular polysaccharides was got when maltose was used as the carbon source.The highest biomass,intracellular polysaccharide and extracellular polysaccharide content were got when yeast extract was used as the nitrogen source.The highest biomass and intracellular polysaccharide content was got at pH=7,and the exopolysaccharide content is highest at pH=5.(2)The c DNAs from different carbon and nitrogen sources,different pH,different developmental stages,and different varieties of L.edodes were used to analyze 10 candidate reference genes by RT-q RCR.Three foftware: Ge Norm,Norm Finder,and Best Keeper were used to analyze the stability,by inputing the Cq value form RT-q RCR.The results showed that Atu,18 S,Rpl4,18 S,and Rpl2 were identified as the most stable internal reference genes in different varieties,different developmental stages,different carbon-nitrogen sources,different pH,and all samples,followed by Rpl4,Btu,Atu,F-actin,and Tsb,respectively.While the most unstable internal reference genes are Pma,F-actin,F-actin,Rpl4,and Pma,respectively.(3)The transcriptional expression levels of three key enzyme genes(UGP,PGI and PGM)were analyzed by RT-qPCR under different carbon,nitrogen sources and pH values.The results showed that the expression levels of the three genes were different,indicating that they may play important roles in the lentinan biosynthesis process.Overall,the relative expression levels of UGP were highest in all samples.When starch was used as the carbon source,all three genes showed relative highe expression level.The relative expression levels of UGP and PGI was highest when yeast extract was used as the nitrogen source.The transcription level of UGP was not significantly different,besides pH=6.5.There was no difference of PGM expression under all pH conditions.The highest transcriptional expression levels of PGI were detected under pH 6.0 and 6.5 samples,which was 4.4 and 4.8 times to pH=7.0 sample.(4)The highest content of lentinan and transcript expression levels of three key enzyme genes were observed when starch was used as the carbon source.When yeast extract was used as the nitrogen source,the content of intracellular and extracellular polysaccharide was the highest,and the transcript expression levels of UGP and PGI were the highest.The intracellular polysaccharide content increased with the increase of pH value,but the extracellular polysaccharide content was decresed.There was no obvious regularity of transcript expression levels of the three key enzyme genes.There is no significant correlation between the transcriptexpression levels of the three key enzyme genes and the polysaccharide content,indicating the complexity of polysaccharide metabolic pathways,the translation and modification of after transcription.