Comparative Transcriptome Analysis Of Samsoniella Hepiali Responses Of Salicylic Acid Induction And Signal Transduction Research

Cordyceps acid has been reported to have bioactivities including antioxidant,anti-inflammatory,and immune regulation,and is one of the important quality factors that directly affect cordyceps fermentation.However,the content of cordyceps acid in conventional fermentation is low.Salicylic acid（SA）can induce the expression of specific genes and promote the synthesis and accumulation of metabolites by triggering the biological self-defense mechanism.Taking the SA-induced regulation of the secondary metabolites of Samsoniella hepiali（S.hepiali）as the foothold,this thesis focuses on the optimal conditions for SA-induced cordyceps acid anabolism,carries out transcriptome sequencing,and explores the SA induction mechanism of the anabolic response of cordyceps acid in S.hepiali by comparing transcriptomics methods.The main conclusions are as follows:1.The effect of SA on hyphae growth of S.hepiali was not significant.It could promote the accumulation of cordyceps acid,and there was a concentration effect on the content of cordyceps acid.In the range of 0-200μM,200μM SA induced the most obvious effect,reaching 13.1 mg/g,which was 1.84times of the control group.In addition,in different adding time experiment,it was found that the induction effect of SA on cordyceps acid was the most obvious on the 3rd day.In conclusion,the optimal induction condition of SA was 200μM on the 3rd day.2.Under the optimal induction conditions,the content of cordyceps acid was the highest when SA was treated for 8 h.After treating for 8 h,high-throughput transcriptome sequencing was performed for induced and uninduced mycelia.A total of 40.37 Gb of Clean data was obtained,and 13 592 Unigene were annotated.Combined with the KEGG database and reference,we constructed a synthetic and metabolic pathway of cordycepic acid in S.hepiali and speculated a new synthetic and complementary pathway.A total of 2 574 DEGs were obtained by transcriptome sequencing,among which 1 135 were up-regulated and 1 439 were down-regulated.Annotation classification and enrichment analysis showed that these DEGs were mainly concentrated in catalytic activity,metabolic synthesis,metal ion signal transduction,defense mechanism and other biological processes.Further analysis showed some key enzymes in Ca2+signaling pathway and SAR signaling pathway play important roles in response to SA regulation.And co-expression analysis found that glk,gpi,gla,mpi,mtl D,mdh,pgm,ugp2,ugdh,uxs,rpib,hk,and hxpb were key enzyme genes regulating the metabolic pathway of cordyceps acid.3.Transcriptome and q-PCR quantitative results showed that the expression of key enzyme genes related to cordyceps acid synthesis was significantly increased after SA treatment,while the expression levels of key enzymes in glucose metabolism pathway were decreased.These results indicate that SA enhances the key enzymes in the metabolic pathway of cordyceps acid synthesis and inhibits the glucose metabolic pathway,thus triggering metabolic flux to achieve the synthesis regulation of cordyceps acid.Furthermore,promoter prediction analysis showed that there were SA response elements,hormone action elements and MYB transcription factor action elements in the promoter of the key enzyme genes of the cordycepic acid synthesis pathway,indicating that SA promoted the expression of cordycepic acid pathway genes through the hormone signaling pathway and MYB transcription factors.4.The results of laser confocal microscopic observation of Intracellular Ca²⁺in S.hepiali induced by SA showed that SA treatment could improve intracellular Ca²⁺content,and the results of q-PCR and transcriptomic sequencing showed that SA promoted the transcription expression of Ca²⁺signaling pathway genes.The co-treatment of Ca²⁺signaling pathway inhibitors EGTA,La Cl₃ and W7 with SA can inhibit the expression of related genes and reduce the accumulation of cordycepic acid.Under the co-treatment of EGTA and SA,exogenous Ca Cl₂can weaken the inhibition effect of EGTA to a certain extent,indicating that under the stress of SA,Ca²⁺is involved in the regulation of the anabolism of Cordyceps acid.In addition,the expression levels of NPR1 protein and BTB/POZ protein in SAR pathway increased under SA treatment,while the expression levels of PAL decreased,suggesting that SAR signaling pathway is also involved in regulating cordyceps acid metabolism in S.hepiali.Therefore,Ca2+and SAR signaling pathways mediate the regulatory effects of SA on the metabolism of Cordyceps acid.