Study On Genetic Developmental Characteristics,polysaccharide Fermentation Regulation And Its Neuroprotective Efficacy Of Agaricus Sinodeliciosus Var. Chaidam

Agaricus sinodeliciosus var.Chaidam is a rare wild and underground edible mushroom growing in the Qinghai-Tibet Plateau,China.Faced with such problems as depletion of wild resources,unclear genetic background and unexplored physiological active substances,this unique and rare resource has not been effectively exploited and utilized.In this thesis,with the help of nucleic acid technology including whole genome and transcriptomics,the gene function,species evolution and phylogeny of this species were analyzed comprehensively and deeply,which laid a foundation for the research of the key physiological active substances of this species and the development of high-quality seed resources.Combined with the genetic characteristics of lignocellulosic degradation and synthesis of various functional components,biomass was used as substrate fermentation to realize the synthesis of high value-added active substances with low cost substrates.The synthesis of exopolysaccharide（EPS）was promoted by means of fermentation metabolism regulation and oxidative stress induced by ultrasonic.Then,EPS was isolated,purified and analyzed,and its effect and mechanism in improving Alzheimer’s disease were studied through cell and in vivo animal experiments.The main results are summarized as follows:1.The high-quality chromosomal level gene assembly map of A.sinodeliciosus var.Chaidam was completed for the first time.The genome sequence size of this macrofungal strain was 36.37 Mb,N50 was 2.8 Mb,and it was mounted on 12 pseudochromosomes.The strain is rich in hydrolase genes that degrade cellulose,hemicellulose,lignin,pectin,chitin and starch.Through the analysis of metabolic product synthesis pathway,the species contained the skeleton genes for the synthesis of terpenoids,ergosterols and polysaccharides,which were typical secondary metabolites of edible macrofungus.It has the potential to synthesize various active substances and degrade lignocellulose.2.Combined with the growth characteristics and physiological habits of this species,comparative genomic analysis was performed with 12 species.The estimated differentiation time between this species and its related species is about 1.6 Mya.The results of gene family cluster analysis showed that five genes were common to the underground mushroom,which were mainly involved in heterocyclic compound binding,DNA repair and binding,meiosis,mitochondrial reverse transcriptase.Expansion and contraction analysis of gene families showed that the expanded genes were mainly concentrated in amino acids,secondary metabolites,antibiotic biosynthesis,glycolysis and gluconeogenesis,and HIF-1 signaling pathways.The contracted genes are mainly involved in regulating protein output.In addition,positive selection analysis showed that protein processing and cell response to stimuli were regulated by positive selection during species evolution.3.To elucidate the developmental regulation mechanism of this species,we successfully achieved uncovered bag culture of this species,obtained the primordia and mature fruiting bodies,and conducted transcriptome analysis of different developmental stages of this species.The results of different-expressed genes（DEGs）showed that DEGs were most abundant in the promordium stage of mycelia development.Transcription factors and hydrophobic proteins can regulate the development of fruite bodies.Hydrophobic proteins ABH1,ABH3 and SC3 are significantly up-regulated in primordium formation and primordium development into fruiting bodies respectively to jointly promote fruiting body development.Environmental signaling factors together induce the development of edible macrofungus.The genes related to lignocellulosic utilization were also differentially expressed at different development stages.4.Macrofungus can secrete lignocellulosic degrading enzymes to transform agricultural waste into high-value active substances.However,for the edible macrofungus with poor lignin degradation ability,the stubborn lignin structure on the biomass surface hindered the release of carbon sources.Therefore,we established a strategy of efficient synthesis of active substances using bagasse lignocellulose as substrate for A.sinodeliciosus var.Chaidam.Chemical methods combined with ultrasonic pretreatment were used to strengthen the delignification process,and the chemical composition,crystallinity,surface lignin and chemical structure of bagasse were characterized.The results show that Fenton combined with ultrasound-assisted Na OH pretreatment has the best delignification effect,with the cellulose content increased by 106.45%,while hemicellulost and lignin decreased by 48.34%and 74.15%,respectively.The biomass,intracellular polysaccharides,total phenols and terpenoids of the strain were 2.4 times,1.46times,1.36 times and 1.66 times that of the non-delignification group,respectively.The established fermentation kinetics model can well fit and predict the growth of bacteria,the secretion of enzymes related to substrate degradation and the synthesis of active substances.This study provides insight into the transformation of low-cost lignocellulose into high-value functional compounds for edible macrofungus.5.A.sinodeliciosus var.Chaidam is a rare edible macrofungus with various biological characteristics,especially its EPS production.However,low yield limited the development and application of edible macrofungus and EPS.Therefore,it is very important to construct positive and effective cultivation strategies.To this end,we constructed an efficient ultrasonic stimulation strategy under liquid culture conditions to promote mycelia growth and EPS biosynthesis.The optimization of ultrasonic stimulation conditions showed that48 h fermentation for 5 min of ultrasonic treatment can significantly improve EPS production and stimulate mycelial growth,compared with the control group,by 26%and15.03%,respectively.In addition,ultrasonic stimulation can increase the permeability of cell membrane,change the structure of mycelia,and reduce the diameter of mycelia,so as to promote mass transfer and thus increase EPS and mycelia production.6.The EPS-2 was isolated and purified from the fermentation broth of A.sinodeliciosus var.Chaidam with an average molecular weight of 18 k Da.Structural characterization showed that EPS-2 was composed of mannose,galactose and a small amount of rhamnose,glucose,fucose,ribose,glucuronic acid and galactosamine.The glycosidic bond structure and NMR analysis showed that the main chain of EPS-2 was→1)-D-Galp-（6→,→1）-D-manp-（2,6→,→1）-D-galp-（2→and→1）-t-manp,with multiple branches.EPS-2 can inhibit the formation of Aβ_1-42 oligomer and reshape the conformational transition of Aβ_1-42 oligomer,thereby reducing the cytotoxicity and oxidative stress of SH-SY5Y induced by Aβ_1-42 oligomer.In conclusion,EPS-2 can block the accumulation of Aβ_1-42 and reduce its neurotoxicity.7.The influence of EPS on AD in vivo was deeply studied.AD rats induced by Aβ_1-42 were given EPS gavage for 21 days to detect the effects of EPS on cognitive ability,oxidative damage indexes and inflammatory factors of AD rats,and investigate the specific mechanism combined with gut microbiota and non-targeted metabolomics.The results showed that 200 mpk EPS could significantly improve the cognitive impairment and relieve anxiety behavior in AD rats.EPS significantly reduced the level of lipid oxidation and inflammatory cytokines TNF-αand IL-1βin the brain tissue of AD rats,mediateed the m TOR-HIF-1αpathway to enhance phagocytosis of Aβ_1-42 by microglia.EPS intervention can inhibit neuroinflammation,oxidative damage and improve cognitive impairment by remodeling gut microbiota in AD rats.In addition,EPS treatment changed the intestinal microbial metabolic profile of AD rats induced by Aβ_1-42,and increased the metabolic product calcitriol by inhibiting Turicibacter,thus playing a key role of anti-AD.