| China is the largest producing country of edible and medicinal fungi in the world. Antrodia cinnamomea (A. cinnamomea), which is famous in Taiwan, is rich in triterpenoids, adenosine, polysaccharides and other active substances and with the function of enhancing immunity, hypoglycemic, anti-allergic, protecting liver. To understand the forming mechanisms of A. cinnamomea and material differences in metabolic pathway, in order to provide a theoretical basis for further exploration of triterpenoid biosynthesis. Building the genetic transformation system, optimization of mycelium liquid fermentation medium, genes cloning and gene expression analysis of MVA biosynthesis in mycelium and fruiting bodies, transcriptomics and metabolomics on A. cinnamomea were carried out in this study. The main conclusions were as follow:Firstly, the growth rates of A. cinnamomea mycelium were increased firstly and decreased afterwards in the solid and liquid medium. The biomass reached to the highest after culturing for 21-28 d. The mycelia was collected after culture for 14 d and then dissolved by 10 mg·mL-1 lywallzyme in 30℃ for 4 h, the protoplast yield was 3.55×105·ml/1, the regeneration rate of protoplast was 4.59% in TB3 medium. The protoplast of A. cinnamomea could be mediated transformation by PEG-CaCl2 under the concentration from 20-40%. The transformation efficiency was the highest under PEG concentration in 40%. The exogenous gene was integrated into the A. cinnamomea genome, which was confirmed by fluorescent microscope and PCR on the transformats containing GFP tag and hygromycin resistance.Secondly, the mycelium biomass medium and triterpenoid-rich on liquid culture of A. cinnamomea were screened by the experiments of Plackett-Bruman, the steepest ascentt and response surface. The results showed that mycelium biomass optimum medium contents of A. cinnamomea were 35.16 g·L-1 glucose,42.86 g·L-1 maltose, 20.80 g·L-1 yeast extract,0.39 g·L-1 VB,,0.85 g·L-1 chitosan sugar. The mycelium biomass was 1.6765± 0.0103 g·100 mL-1 (DW) after culturing for 15 days at 28℃ on a shaker at 120 rpm·min-1. The mycelium triterpenoid-rich optimum medium contents of A. cinnamomea were 10.32 g·L-1 glucose,42.58 g·L-1 maltose,15.37 g·L-1 peptone,1.12 g·L-1 MgSO4,0.63 g·L-1 KH2PO4,0.55 g·L-1 JUNCAO Ganoderma lucidum substrate water extracts (JCGLWE). The mycelium triterpenoid content was 34.03±1.264 mg·g-1 (DW) after culturing for 15 days at 28℃ on a shaker at 120 rpm·min-1.Thirdly, pyrophosphate decarboxylase (mvd) and squalene epoxidase (se) were amplified by RACE using degenerate primers. The full-length of mvd and se gene were 1268 bp and 1607 bp, which including 1 and 3 introns and encoded 402 and 481 amino acids, respectively. The expression levels of mvd and se of A. cinnamomea under different stages were analyzed by real-time quantitative PCR (qRT-PCR). The results showed that expression levels of mvd and se in mycelium were the highest after culturing for 7 days. The expression levels of mvd and se in mycelium were decreased as extension of culture time and were stable after culture for 28～42 days. The triterpenoids content in mycelium was gradually increased as culture time less than 28 d, and was the highest (39.192±2.025 mg·g-1) after culturing for 28 days, which was second only to the triterpenoid content in the fruiting bodies of A. cinnamomea (49.391±2.675 mg·g-1). The triterpenoids content in mycelium was decreased as the culture time more than 28 d. The triterpenoids content of A. cinnamomea in fruiting body was lowest (11.530±0.733 mg·g-1) cultivated by corncob. The differences of triterpenoid content between each sample and the control group were significant (P< 0.05).Fourthly, the transcriptome GO and KEGG were determined on the fruiting bodies of A. cinnamomea (Sample 1), mycelium in liquid culture for 14 days (sample 2) and 28 days (Sample 3). The results showed that there were 324 differentially expressed genes between sample 1 and 2 by the gene expression analysis, inculdig 307 down-regulated genes and 17 up-regulated genes, which were enriched in 83 GO-term. There were 271 differentially expressed genes between sample 1 and 3, including 245 down-regulated genes and 26 up-regulated genes, wchih were enriched in 78 GO-term. There were 337 differentially expressed genes between sample 2 and 3, including 244 up-regulated genes and 93 down-regulated genes, which were enriched in 46 GO-term.The main differences of metabolic pathways between fruiting bodies and mycelia were focused on carbon metabolism, oxidative phosphorylation, Krebs cycle, glycolytic solution, amino acid biosynthesis. The differences of metabolic pathways in mycelium were mainly in fatty acid metabolism, NOD receptor signaling pathway, PPAR signaling pathways. The results showed that the carbon metabolism and the amino acid metabolism in different forms of A. cinnamomea were abnormal. The fatty acid metabolism and varieties of different signaling pathways during culture time of mycelium were disorder, and the change of fatty acid metabolism was the most obvious. Two stable expression gene s were as housekeeping gene by screening out of the transcriptome sequencing. The differentially expressed genes during the triterpenoid pathway were verification by the housekeeping gene.Fifthly, metabolome analysis of the fruiting bodies of A. cinnamomea (Sample 1), mycelium in liquid culture for 14 days (sample 2) and 28 days (Sample 3) were carried out in this study, it showed in the results that 21 and 26 kinds of metabolic differences were identified between sample 1 and 2 under positive and negative ions condition, respectively.14 and 28 kinds ofmetabolic differences were identified between sample 1 and 3 under positive and negative ions condition, respectively.20 and 29 kinds of metabolic differences were identified between sample 2 and 3 under positive and negative ions condition, respectively. Meanwhile, the main metabolic differences pathway between fruiting bodies and mycelia were including amino acid synthesis metabolism, carbon metabolism and glucose metabolism, the common metabolites differences were 47 in total. The metabolic differences pathways in mycelium were including amino acid synthesis and fatty acid metabolism, the common metabolites differences were 43 in total. The specific potential biomarkers between fruiting bodies and mycelia under negative ion conditions were Coproporphyrin Ⅲ, NG, NG-dimethyl-L-arginine, imidazole, UDP-N-acetylglucosamine, uridine diphosphate glucose, ornithine. The specific potential biomarkers between different cultured time of mycelia were D-galactose, glycosides amino acids, P-D-fructose-2-phosphate.Above all, biosynthesis-related research and comprehensive discussion on A. cinnamomea triterpenoid biosynthesis pathway were carried out around medium screening, gene cloning and expression, transcriptome and metabolome analysis. The results provided scientific basis and theoretical foundation laid a good foundatio for further excavation of A. cinnamomea triterpenoids biosynthetic gene, and also laid a good foundation for industrial production of A.cinnamomea mycelium. |
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