Study On The Pathway And Key Genes Of Intracellular Polysaccharide Anabolism From Sanghuangporous Sanghuang By ARTP Mutation Based On Multi-omics Analysis

Sanghuangporous sanghuang,a rare medicinal fungus,belongs to Basidiomycota,Agaricomycetes,Hymenochaetales,Hymenochaetaceae and Sanghuangporus.It usually grows on Morus L.in central and South China and has been proved to exert obvious anti-tumor,immune enhancing,anti-diabetics,anti-oxidant,lowering serum uric,anti-inflammatory and other pharmacological effects.S.sanghuang has gradually become a new bright spot in the research field of edible fungi.Polysaccharides are the main bioactive substance of S.sanghuang,which has obvious pharmacological effects such as lowering blood sugar,lowering uric acid,antioxidation and regulating immunity.High-yield polysaccharide strain is a prerequisite for to artificial cultivation of S.sanghuang.A high-yield polysaccharide strain A130 was obtained by ARTP mutation in our laboratory.The effects on physicochemical properties and biological activities of intracellular polysaccharide from S.sanghuang by ARTP mutation were studied in this paper.The changes of pathway and key genes of intracellular polysaccharide anabolism from S.sanghuang by ARTP mutation were investigated based on multi-omics analysis.It is of great significance to reveal the mechanism of high-yield-polysaccharide strain by ARTP technology.This paper is divided into five parts,the main results are as follows:1.The effects on physicochemical properties and biological activities of intracellular polysaccharide from S.sanghuang by ARTP mutationSix intracellular polysaccharides were obtained by liquid fermentation of the wild strain(SH1)and the mutant strain(A130).The mycelium was lyophilized and weighed.The polysaccharides were extracted by water and precipitated by gradient alcohol.They were named SH1-20,A130-20,SH1-50,A130-50,SH1-70 and A130-70,respectively.The physicochemical properties and in vitro immune activity of six polysaccharide fractions were compared.The results showed that the biomass of A130 was 15.80g/L,14.43%higher than that of SH1,and the polysaccharide content of 0.78g/L,27.86%higher than that of SH1.The molecular weight of A130-20,20%alcohol precipitated polysaccharide component,was higher than that of SH1-20,but the molecular weight of 50%and 70%polysaccharide components were similar.The monosaccharide compositions of the mycelia polysaccharide of S.sanghuang by ARTP mutation were significantly different from that of the original strain.At the same elution time,the molecular weight of A130-20 is larger than that of SH1-20,but the particle size is smaller than that of SH1-20,which shows that the structure of A130-20 is more closely aligned.According to the RMS comformation map,A130-20 is more seriously pendulous,which suggested that A130-20 had highly branched and closed molecular structure.The immune activity of polysaccharide A130-20 was greatly improved by ARTP mutagenesis compared with SH1-20.It can be inferred that macromolecule?-glucan contributed to better immune activity.The new polysaccharide A130-20-P1 was isolated and purified from A130.The structure analysis showed that A130-20-P1 was a large molecule of?-glucan with a molecular weight of 1.210×10~7 Da.The connection modes of glucose residues were end base connection,1,3-connection and 1,3,4-connection,respectively.The molar ratios were 1.0:2.1:1.1.The primary structural repeating unit of A130-20-P1 was listed as follows:2.Whole genome sequencingof S.sanghuang(SH1)The third generation of single molecule sequencing technology was used to sequence the whole genome of SH1 and 106 contigs were finally assembled.The largest contig in the assembled sequence was 5607247 bp,and the length of 50%of the total length of contig is 1053625 bp;the total length of all the assembled contigs is 34164286bp;the GC content of the assembled contig is 47.87%.Through KEGG database,339genes were predicted to participate in carbohydrate synthesis pathway,38 genes were related to glucan synthesis pathway.3.Study on the pathway and key genes of intracellular polysaccharide anabolism from S.sanghuang by ARTP mutation based on transcriptomeIn this study,6 samples were sequenced by reference transcriptome,and 45.12G of Clean Data was obtained.The average GC content was 48.09%.Through transcriptome analysis,31 differentially expressed genes were obtained by transcriptome analysis of A130 VS SH1,of which 21 were up-regulated and 10 were down-regulated.The enrichment analysis of 31 differentially expressed genes in KEGG database was carried out to two metabolic pathways.One was a sugar metabolism pathway,ko 00500,which was down-regulated by CBH1 gene.A gene repair related pathway,ko 03430,was excavated and upregulated to MSH6 gene.Real-time fluorescence quantitative PCR was used to verify the genes of two key enzymes,CBH1 and MSH6.The results showed that the difference multiples of CBH1 gene in A130 VS SH1 and MSH6 were 0.45 times and6.3 times respectively,which were consistent with the results of transcriptome.Compared with SH1-20,the ratio of glucose was increased,the CBH1 gene was down-regulated,and the activity of?-1,4-glucan hydrolase(EC 3.2.1.91)was decreased.After ARTP mutation,it can be found that?-glucan A130-20-P1 isolated from A130-20 has?-1,4 glucoside bonds in the branch chains.Polysaccharide synthesis is a dynamic process of synthesis and decomposition.Therefore,the down-regulation of CBH1 gene inhibited the hydrolysis of?-glucan,and the isolation of components A130-20 resulted in?-glucan A130-20-P1.The branch chains were in the form of?-1,4glucoside bonds,and the results were consistent.MSH6 is a DNA mismatch repair gene.S.sanghuang strain was affected by high-energy active particles of plasma in the mutagenesis process of ARTP,causing DNA damage,high mutation rate and abundant mutation sites.In the process of DNA self-repair,the SOS repair mechanism induced by biological cells brings about a variety of mismatches,and these mismatches may change the genes controlling the enzyme proteins related to polysaccharide synthesis,thus promoting the synthesis of polysaccharides and the structural changes of polysaccharides.4.Study on the pathway and key enzyme of intracellular polysaccharide anabolism from S.sanghuang by ARTP mutation based on i TRAQThe differential proteomics of A130 and SH1 was studied by i TRAQ combined with two-dimensional liquid chromatography.A total of 2,274 proteins were identified and 2,064 were screened as trusted proteins.Based on the screened trusted proteins,69differentially expressed genes were obtained by analysis of the A130 VS SH1 protein group,among which 23 were up-regulated and 46 were down-regulated.The enrichment analysis of 69 differentially expressed proteins in KEGG database was carried out,and 23 metabolic pathways were found,one of which was a glucose metabolism pathway,ko 00500,which was consistent with the different genes excavated from the genome.The pathway identified three proteins related to glucose metabolism,TREH,tre A,and tre F.In this glucose metabolism pathway,the expression of?-trehalase in A130 was lower than that in SH1.It promotes the formation of trehalose,and acts as a source of energy at the initial stage of fungal spore germination,thus promoting the synthesis of polysaccharide from S.sanghuang.At the same time,trehalose,as an important stress protective substance against environmental stress,exists a variety of synthetic and catabolic pathways in different organisms.The synthesis,decomposition and regulation of trehalose are important mechanisms of bioresistance.5.Combined analysis of differential genes and proteinsCombined analysis of differential proteins and differential genes was performed to enrich 1 metabolic pathway related to glucose metabolism.In the biosynthetic pathway of ko00130 ubiquinone and other terpenoid quinones,the expression of NADPH dehydrogenase(EC 1.6.5.2)increased in the mutated strain A130.NADPH dehydrogenase is an important oxidoreductase involved in various physiological processes and biochemical metabolism in organisms.It can influence the thermodynamic driving force of various reactions in vivo by regulating the content of NADP(H),a key component of REDOX metabolism in cells,and thus promote the synthesis of polysaccharides.