Cloning And Functional Analysis Of Positive Regulators GlbHLH1 And GlbHLH5 For Triterpenoid Biosynthesis In Ganoderma Lingzhi

Ganoderma lingzhi is a basidiomycete fungus belonging to the genus Ganoderma,which is rich in triterpenoids,sterols,adenosine and other active substances.Triterpenoids are the key active components of Ganoderma lingzhi,and have many pharmacological effects such as anti-inflammatory,antiviral,antitumor.However,as a kind of secondary metabolites,the triterpenoid content in Ganoderma lingzhi fruiting bodies and mycelia is extremely low,which limits its efficient utilization.Therefore,it is necessary to reveal the metabolic regulation mechanism of triterpenoids in Ganoderma lingzhi by molecular biology methods,and then break through the bottleneck problem of low biosynthesis efficiency through precise metabolic regulation.Transcriptional regulation is an important aspect of fungal metabolism regulation.Using transcription factors to regulate metabolism has more advantageous than simply modifying one or more synthesis pathway genes,but there are few studies in Ganoderma lingzhi.In this study,genome mining,gene expression correlation analysis,subcellular localization,transcriptional activation activity analysis,gene overexpression and silencing were used to identify bHLH transcription factors that effectively promote triterpenoid biosynthesis in Ganoderma lingzhi.It is not only beneficial to carry out precise metabolic regulation aiming at improving triterpenoid content in Ganoderma lingzhi,but also provides theoretical evidence for revealing the transcriptional regulation mechanism of triterpenoid biosynthesis in Ganoderma lingzhi.The main research results are as follows:1.Conserved domain search was used to identify and analyze the entire genome protein sequence domain of Ganoderma lingzhi.After deleting the redundancy,13 complete bHLH families transcription factors were obtained,named GlbHLH1～GlbHLH13.Phylogenetic tree analysis showed that 13 bHLH proteins were closely related to Panax verus and Lentinula edodes.Promoter analysis showed that the promoter region of all Ganoderma bHLH genes contained Me JA responsive elements.Exogenous Me JA significantly promoted the accumulation of triterpenoids and significantly increased the expression of HMGR,SQS and LS,and GlbHLH1 and GlbHLH5 also responded to the induction of Me JA.A pearson correlation analysis showed that GlbHLH1 and GlbHLH5 were positively correlated with the key genes of triterpenoid biosynthesis in Ganoderma lingzhi(P<0.01).Therefore,GlbHLH1 and GlbHLH5 are candidate genes for regulating triterpenoid biosynthesis induced by Me JA.2.The CDS sequences of GlbHLH1 and GlbHLH5 were cloned,and the subcellular localization vector p BARGPEI-GlbHLH1/GlbHLH5-GFP and yeast transcriptional activation vector p BDGlbHLH1/GlbHLH5 were constructed.The subcellular localization vector was transformed into Ganoderma lingzhi and found that the recombinant strain had obvious fluorescence in the nucleus,while the control strain had no fluorescence,indicating that GlbHLH1 and GlbHLH5 proteins were located in the nucleus of Ganoderma lingzhi.The transcriptional activation vector was transformed into yeast AH109,and the results showed that the p BD-GlbHLH1/GlbHLH5 transformants grew well on the medium supplemented with 3-AT SD/-Trp/-His/-Ade,while the control strain did not grow,indicating that both GlbHLH1 and GlbHLH5 had transcriptional activation activities,so GlbHLH1 and GlbHLH5 had the basic characteristics of bHLH activity.Promoter analysis showed that the promoter regions of the key enzyme genes HMGR,SQS and LS in ganoderma triterpenoid biosynthesis pathway contained the binding site G-box of bHLH,so GlbHLH1 and GlbHLH5 may activate the transcription of HMGR,SQS and LS to improve the synthesis of ganoderma triterpenoids.3.By homologous recombination,p BARGPEI-GlbHLH1 and p BARGPEI-GlbHLH5 overexpression vectors were successfully constructed and transformed into Ganoderma lingzhi protoplasts to obtain stable over-expression transgenic strains.The determination of triterpenoid content and the expression analysis of key enzyme genes showed that compared with the control strain,the triterpenoid content of the three OE-GlbHLH1 overexpression strains increased by 25%,22% and 38%,respectively(P<0.05),and the relative expression levels of HMGR,SQS and LS of GlbHLH1 and key enzyme genes for triterpenoid biosynthesis increased significantly(P<0.05).The triterpenoid content of the three OEGlbHLH5 overexpression strains increased by 45%,37% and 21%,respectively(P<0.05).The relative expression levels of GlbHLH5 and key enzyme genes HMGR,SQS and LS increased significantly(P<0.05).The transcription and expression trends of key triterpenoid biosynthesis genes in GlbHLH1 and GlbHLH5 overexpression strains were consistent.4.The p Silent-1-GlbHLH1 and p Silent-1-GlbHLH5 silencing vectors were successfully constructed by homologous recombination and enzyme digestion,and the stable transgenic strains were obtained by transforming Ganoderma lingzhi protoplasts.The analysis of triterpenoids content and the expression of key enzyme genes showed that compared with the control strain,the triterpenoid content of the two GlbHLH1 silencing strains was significantly reduced by about 15% and 30%,respectively.The relative expression levels of GlbHLH1 and key enzyme genes HMGR,SQS and LS were significantly reduced(P<0.05).The triterpenoid contents of the three Si-GlbHLH5 silencing strains were significantly reduced by about 24%,31% and 19%,respectively(P<0.05).The relative expression levels of GlbHLH5 and the key enzyme genes of triterpenoid biosynthesis LS,HMGR and SQS were significantly reduced(P<0.05).The transcriptional changes of silencing GlbHLH1 and GlbHLH5 genes were also consistent with the expression trends of key genes in triterpenoid accumulation and biosynthesis pathway in Ganoderma lingzhi.In summary,GlbHLH1 and GlbHLH5 transcription factors are bHLH transcription factors that positively regulate triterpenoids biosynthesis in Ganoderma lingzhi in response to Me JA.In response to JA signal,bHLH transcription factor specifically binds to G-box to regulate the transcription of key genes HMGR,SQS and LS in triterpenoids metabolic pathway of Ganoderma lingzhi,and further promote the biosynthesis of triterpenoids.