| Wolfiporia cocos(Schwein.)Ryvarden&Gilb,belongs to the genus Wolfiporia of the familyPolyporaceae.It is a traditional Chinese medicine(TCM)that is always applied both medicinal and edible and has long medicinal history,which is the dried sclerotia of Polyporaceae fungal W.cocos.Modernresearch have shown that triterpenoid,the active ingredient of W.cocos,have many pharmacological effects.Researches of W.cocos mainly focus on the aspects of medicinal ingredients and pharmacological effects,while few researches on transcriptome and molecular level.It is a hot research topic to improve thebiosynthesis and accumulation of triterpenoid in W.cocos and to select the strains with high triterpenoid production.In present study,through the traditional methods such as optimization of sexual sporulation conditions,the isolation and screening of single spore strains,combined with transcription,metabonomics and othermodern methods,a comprehensive study revealed key genes of W.cocos triterpenoid biosynthesis and its regulation factors,and analyze their relation with triterpenoid,then,the regulatory genes of W.cocostriterpenoid biosynthesis were obtained.It lays a scientific foundation for improving the yield oftriterpenoid and the breeding of strains with high yield of triterpenoid in the future.The main results are as follows:1.Condition optimization of W.cocos sexual sporulationIn order to construct a near isogenic line,obtain different strains with consistent genetic basis anddifferent total content of triterpenoid,this study has optimized the conditions for sexual spore production of W.cocos in laboratory culture.Single factor experiments were carried out on medium,culture container,intial medium p H value,temperature and light conditions.The optimal condition of spore production of W.cocos is(8.12±0.11)×10~9/bottle in the culture bottle of PDA medium A with p H 6.0～7.0,after dark culture of 25～28℃for 7 days and alternating culture of light and dark for 38 days.2.Two single spore strains with significant difference in total triterpenoid content of W.cocos were screened out.In order to avoid material differences in genetic background and different growth stages to influence on the results of this study,two monosporal strains,high yield(H)and low yield(L),were screened out from the 236 sexual reproductive progenies of the same W.cocos 5.78.3.Transcriptome analysis of W.cocos myceliaTwo strains of W.cocos(H&L),three times of culture(cultured mycelia for 17 days,34 days,and 51days),3 times of biological repetition,a total of 16879 genes were assembled from 18 transcripts,of which12260(72.63%)were annotated by NR database.96.51%of the encoded protein genes are known proteins,and 42.96%are proteins with amino acid number greater than 300.SSR shows a significant preference for GC repeat,dominated by trinucleotide repeat unit(CCG/CGG)n.Among the differentially expressed genes between the two groups,FDPS,erg11,erg26,erg6 and SQLE were related to the triterpene synthesispathway,while Pm20d2 and nor A were outside the triterpene synthesis pathway,which were speculated to be related to triterpenoid biosynthesis.In this study,10 core genes(HMGCR,COQ2,ERG2,ACAT,TAT,erg11,erg26,SQLE,CAO2 and FACE1)and 7 related core gene(Pm20d2,nor A,chromatin remodeling ATPase genes,adh,ftmp450-1,unknown proteins Unigene0011374 and unigene0001029)were obtained by transcriptional STEM analysis.Most of the core genes belong to the gene of membrane proteins or sterols and the key components of cell membrane homeostasis.Maintaining high triterpenoid accumulation in W.cocos may also require a stable membrane structure,so the accumulation ability may be related to the high synthesis ability of sterols.According to the results of WGCNA analysis in this study,there were 9 core genes related to thesynthesis and accumulation of triterpenoid,such as ACAT1-b,hgs A,mvd1,SQLE,erg11,TAT,erg26 and two erg6,and outside the pathway there were multiple regulatory factors and protein genes such as Pm20d2and nor A.Their expressions indicated that the synthesis and accumulation of triterpenoid of W.cocos were closely related to the biosynthesis of sterol.The genes of SQLE,TAT,erg26 and erg11 in the relatedpathways of triterpenoid synthesis,as well as Pm20d2 and nor A outside the pathways,showed extremely high correlation and connectivity in both analytical methods.4.Analysis of metabolism of mycelia in W.cocosA total of 8219 positive and negative ions were detected by LC-MS analysis of a total of 36 samples from two strains(H&L),three culture times(cultured mycelia for 17 days,34 days,and 51 days)and 6times of biological repetition,among which 75914 were known substances,accounting for 92.36%.The enrichment results of differential metabolite KEGG confirmed the results of transcriptome analysis,the biosynthesis and accumulation of triterpenoid were closely related to the synthesis of sterols,maintenance of membrane structure and membrane transport function.The results of STEM analysis of metabolitesindicated that the synthesis and accumulation of triterpenoid and polysaccharides in W.cocos were trading off and taking turns.Correlation analysis of differentially expressed genes and differentially expressedmetabolites showed that FDPS,CAO2,erg11(unigene0015621)and Pm20d2 were the regulatory genes confirmed by metabolic analysis.5.Biological information analysis of regulatory genesThe results of biological information analysis for two genes of Pm20d2 and erg11 showed that all of them are not secreted proteins,but have one coding protein with transmembrane domain.Their secondary structure are mainly random coil,and their tertiary structure are very different,which are the coding protein with different structure and function,and they all are that perform functions on the membrane.Phylogenetic relationship analysis of Pm20d2 confirmed the homology analysis results of transcriptomegene sequence;erg11 was a single source,with a certain genetic distance from other strains and uniqueness.6.Analysis on the regulation mechanism of biosynthesis and accumulation of triterpenoidThe expression of key genes indicated that gene regulation of triterpenoid synthesis may occur atvarious levels from transcription to post-translation processing.The gene expression of other branches with common precursors indirectly regulates the synthesis and accumulation of triterpenoid.Regulatory factors outside the pathway of triterpenoid synthesis also play an important role in the biosynthesis of triterpenoid.In conclusion,through the transcriptome and metabolomics analysis,this study found that a regulatory gene erg11(unigene0015621)annotated in the triterpenoid synthesis pathway and a regulatory genePm20d2 out of the pathway,they were closely negatively correlated with.The synthesis and accumulation of triterpenoid are closely related to the biosynthesis of sterols.The gene expression of other branches with common precursors indirectly regulates the synthesis and accumulation of triterpenoid.Maintaining thehigh triterpenoid accumulation of W.cocos may require a stable membrane structure.The synthesis and accumulation of triterpenoid and polysaccharides are trading off and taking turns.The results of this study lay a theoretical foundation for the production of triterpenoid with W.cocos in biological engineering and also provide a theoretical reference for the production of triterpenoid with other materials.The results are of great significance to improve the yield of triterpenoid and breed of triterpenoid strain with high yield. |
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