The Role Of Calcineurin-responsive Zinc Finger Transcription Factor CRZ1 In Regulation Of Ganoderic Acid Biosynthesis In Ganoderma Lucidum

Ganoderma lucidum,a traditional medicinal mushroom,produces various kinds of bioactive ingredient such as ganoderic acids(GAs)and polysaccharides.GAs possesses significant pharmacological including anti-tumor,anti-HIV,and immunomodulatory effects,so it has received extensive attention.Previous studies have found that the production of GAs and the transcription level of calcineurin-responsive zinc finger transcription factor(CRZ1)were regulated by adding Ca²⁺to static liquid cultures of G.lucidum.CRZ1 is considered to be related to the biosynthesis of GAs,however,there is still lack of information regarding how CRZ1 regulate GAs biosynthesis.Therefore,studying regulation of CRZ1 in GAs biosynthesis is helpful to deeper understand of GAs biosynthesis and regulation mechanism.In this study,the complete gene and c DNA sequence of crz1 in G.lucidum was cloned.The complete sequence was found to be 1431 bp and 1077 bp,respectively.Bioinformatics analysis found that its amino acid sequence has high similarity with CRZ1 of other basidiomycetes,the similarity of the more conservative zinc finger domain is higher than 85%.The engineering strains CRZ1^SI and CRZ1^OE was constructed by crz1 gene-silencing and overexpression.To investigate the effects of CRZ1 on the accumulation of GAs in liquid static fermentation,we tested the GAs content when 10 m M Ca²⁺was added to the culture medium.The crz1 gene-silencing significantly reduced the content of GAs,the highest content of total GAs and GA-Mk,GA-T,GA-S,GA-Me were 69.2%,69%,79.5%,83.4%and 75.8%of the wild type(WT)strain,respectively.The overexpression of crz1significantly increased the content of GAs,the highest content of total GAs and GA-Mk,GA-T,GA-S,GA-Me were 1.21,1.10,1.17,1.12 and 1.20-folds higher than the WT strain,respectively.Moreover,the results of q PCR displayed that the transcription levels of fps and ls in CRZ1^SIstrain,two key genes of GAs biosynthetic pathway,were significantly lower than those in the WT strain.The transcription levels of fps and ls in CRZ1^OEstrain were significantly higher than those in the WT strain.The above results indicate that the gene-silencing and overexpression of crz1 significantly affect the accumulation of GAs and the transcription levels of genes which related to GAs synthesis.The regulation mechanism of CRZ1 in GAs biosynthesis has been further studied by subcellular localization and Electrophoretic Mobility Shift Assay(EMSA).The subcellular distribution of CRZ1 was determined using a G.lucidum mutant expressing CRZ1 with a GFP-tag at the C-terminal.Subcellular localization results showed that the fusion protein CRZ1-GFP accumulated in the nucleus of mycelium when Ca²⁺was added to the culture medium.EMSA results displayed that the in vitro binding of CRZ1to probe which designed on the promoter of fps,these binding bands shifted,that proved CRZ1 has a binding effect with the promoter of fps.In conclusion,we found that CRZ1 accumulated in the nucleus of mycelium when Ca²⁺was added,and regulated the transcription level of fps by binding to its promoter,thereby affecting the biosynthesis and accumulation of GAs.Our results were helpful to in-depth study of GAs biosynthesis and regulation mechanism.