Study On High Temperature Stress Of Lentinus Edodes Based On Multi-omics Analysis

Lenlinula edodes is a kind of edible and medicinal fungi,which has the characteristics of variable temperature firmness,when the temperature is higher than 25?,some problems would appear,such as inhibition of mycelia growth,yellowing of mycelia,sporophore's cap open in advance,and the quality becomes worse.Therefore,it's great guidance significance,to study the molecular mechanism of high temperature stress,which affects the growth and development of Lentinula edodes in order to breed the excellent heat-resistant strains,and improve the cultivation conditions.In this study,18 strains of the main variety of Lentinula edodes and the high temperature restorer strain 18N44 by mutagenesis and screened were used as test materials,through the test of phenotypic and physiological indicates of two strains determination,to evaluate the difference of Lentinula edodes strains in response to high temperature stress,combined with internal reference gene screening and test technology of transcriptomics,proteomics and metabolomics,from the transcription,protein and metabolic levels to analyze the molecular expression patterns of high temperature stress response and explored the molecular mechanism of high temperature stress.The main results are as follows:(1)Phenotypic analysis of mycelial under high temperature stressUnder the optimum stress temperature of 37?,when the stress time gradient is 0,4,8,12,18,24 h observed the growth of Lentinula eddoes mycelium is inhibited,and the branches decreased,and formed a "heat stress circle",by measuring the physiological indexes such as relative electrical conductivity,malondialdehyde content,osmotic potential,reactive oxygen species,and antioxidants and other physiological indicators found that strain 18N44 was more sensitive to high temperature stress,however,after 24 hours of heat-shock treatment and high temperature stress relieved,the recovery of growth rate for strain 18 was 0.881 mm/d and strain 18N44 was 1.613 mm/d,which indicated 18N44 had stronger recovery growth ability.(2)Selection of reference gene under high temperature stressUse of the qRT-PCR method,based on the software of geNorm,NormFinder,and Best Keeper,and combined with test verification,TUB gene(?-Tubulin)was identified as the best internal reference gene in 10 candidate reference genes of Lentinula edodes for the analysis of the gene expression,which provided the necessary guidance and corrections basis for qRT-PCR tests of the expression of Lentinula edodes target gene at high temperature stress.(3)Transcriptome sequence analysis under high temperature stressTranscriptome sequences were performed on two strains of Lentinula edodes at various high temperature stress points by uses of the platform of PacBio Sequel and Illumina Hiseq Xten.Combined with the result of full-length transcriptome isoform library with second-generation sequence results,the differentially expressed genes of the two strains were compared at the different time point of high temperature stress,based on functional enrichment and expression trend analysis,the results showed that strain 18N44 was involved in more biological processes related to high temperature stress and responded more actively to high temperatures stress.The function of enrichment mainly focused on the spatial folding of proteins,protein stability,cellular heat adaptation,heat-shock protein binding ability and so on.There are a large number of heat-shock proteins genes such as hsp100,hsp90,hsp70,hsp60 and hydrophobic protein gene hydl differentially expressed in strain 18N44,this heat-shock proteins and hydrophobic proteins can effectively repair denatured proteins under high temperature stress,reduce misfolded proteins,protect the integrity of cells to enhance resistance and reduce the damage,and protect organisms,regulation at RNA level during high temperature stress can effectively protect the body from excessive damage.When the high temperature relieved,could rapid complete the biological processes such as transcription,translation,and modification,in order to achieve faster growth recovery.(4)Sequence of metabolites under high temperature stressGC-MS the technology was used,to detect metabolites and expression changes of two strains of two Lentinula edodes strains under high temperature stress.The enrichment results of differential metabolite Pathway for strain 18N44 showed that,the metabolic changes were significant in the metabolism of glutamic acid,aspartic acid,and alanine;TCA cycle;glutathione metabolism,glycine,serine,and threonine.Most amino acids and carbohydrates enriched in these metabolic Pathways were mainly expressed as up-regulation in strain 18,down-regulation in strain 18N44,or synthesis of strain 18 is higher than strain 18N44.The changes of amino acids and glycols provided a theoretical explanation for the more sensitive phenotypic and physiological characteristics of strain 18N44 in response to high temperatures.(5)Functional verification of targeted quantification and exogenous addition of target metabolitesThe trend of relative content,change is basically the similar with the screening of targeted metabolite content determination and non-targeted screening identification,which proved the accuracy of non-target identification results.And through the test by adding exogenous materials vitrificated the function of trehalas and mannitol as key target metabolites,the results showed that the two metabolites not only had the nutritional function of promoting growth under normal conditions,can also effectively protect the body from excessive damage and enhance the ability of body repair under high temperature stress,and demonstration of a verified new method proved reliability of metabonomic screen,which conducive to improved cultivation measures,optimized batch preparation and preventing high temperature damage.(6)Multi-omics analysis under high temperature stressThe integrative analysis of genes,proteins,and metabolites with significant differences between the two strains under high-temperature stress for 24 hours was carried out,constructed the network map of multi-molecular regulation and Pathway interaction.The results showed that there were 543 genes,20 proteins and 162 metabolites with significant differences after high-temperature stress mainly involved in the signaling Pathways,such as TCA cycle,pyruvate metabolism,propionic acid metabolism,oxidative phosphorylation,carbon metabolism,aminoacyl-tRNA biosynthesis,amino acid biosynthesis,secondary metabolites biosynthesis,pentose phosphate Pathway,glycolysis and gluconeogenesis.These Pathways contained specific regulatory information,which explained the molecular mechanism of strong thermal resilience for strain 18N44,and provided the basis for molecular breeding of high-temperature-resistant strains.The above results preliminarily revealed phenotypic differences and molecular regulation mechanisms of different temperature tolerance Lentinula edodes strains 18 and 18N44 in response to high temperature stress,and predicted the recovery mechanism induced high temperature restorer strain 18N44.However,the regulation mechanism of the recovery stage needs further study,the role of key Pathways needs further test analysis,and the function of exogenous application of target metabolites needs to be further validated during of fruiting body development.