Regulation Of Ganoderic Acids Biosynthesis By Calcineurin In Ganoderma Lucidum

Ganoderic acids（GAs）,a kind of triterpenoids,is one of the most important bioactive compounds of Ganoderma lucidum.GAs has a variety of pharmacological functions,including anti-tumor,anti-HIV,anti-oxidation,and inhibition of cholesterol synthesis.However,the low yield of GA has become an important factor for limiting its commercial application.Research on the regulation mechanism of GAs has become a focus in recent years.Previous study found that the yield of GAs could be improved by addition of calcium ion.Whereas it is still not clear whether calcineurin is involved in the regulation of Ca²⁺on the biosynthesis of GAs.Regulation of GAs biosynthesis by calcineurin requires further research.In this study,we first cloned two genes encoding the catalytic subunit of calcineurin,named cna1 and cna2,respectively.The entire gene of cna1 is 2680 bp and has an open reading frame of 2007 bp.The entire gene of cna2 is 2645 bp and has an open reading frame of 1971 bp.The expression levels of cna1 and cna2 were determined through q RT-PCR under the addition of calcium ion.We found that the transcription level of cna1 and cna2 under the addition of Ca²⁺were 1.6 and 2.5 times higher than those in control group.The results suggested that calcineurin may involve in the biosynthesis of GAs.Subsequently,the effect of calcineurin on the biosynthesis of GA was investigated by inhibiting the function of calcineurin.We silenced cna1 and cna2 gene and determined the content of GA in cna1-silenced or cna2-silenced transformants,respectively.We found that the content of GA has not changed in these silent strains.This might be due to the function of cna1and cna2 are redundant in G.lucidum.Therefore,we inhibited the function of calcineurin by addition of cyclosporine A（Cs A）,an immunosuppressant.The content of GA was determined under the addition of Cs A.The production of total GA and four individual GAs Mk,T,S and Me are 3.33±0.12 mg,175.886±2.56,236.163±3.82,165.239±5.75 and 106.733±2.23μg per100 mg dry cell weight,respectively,which are about 0.67,0.47,0.49,0.21 and 0.74 times than those in the control group.The intermediate metabolites squalene and lanosterol decreased by0.24 and 0.22,respectively.The expression levels of FPS,SQS and LS related to the GA biosynthesis were 0.16,0.3 and 0.46 times than those of the control,respectively.In addition,the effect of calcineurin on the post-translational modification and cellular localization to calcineurin-responsive zinc finger transcription factor（crz1）was studied by inhibiting the function of calcineurin.We obtained the crz1-flag overexpression transformants.The electrophoretic mobility of Crz1 protein was detected by Western blot under different culture conditions.The amount of phosphorylated Crz1 was increased in protein extracts,treated with Cs A.Furthermore,we examined the subcellular localization of Crz1 by fluorescence microscope in crz1-gfp overexpressing strains.In the presence of Ca²⁺,Crz1 was localized into the nucleus.However,the nuclear localization was inhibited under the addition of Cs A.The Crz1 was mostly localized in the cytosol.These results indicated that calcineurin can mediate the biosynthesis of GAs by regulating the activity of Crz1.In summary,we found that calcineurin is involved in the GA biosynthesis under the regulation of Ca²⁺.Calcineurin can regulate the expression of genes related to the biosynthesis of GA by dephosphorylating the transcription factor Crz1,and subsequently regulate the biosynthesis of GAs.Studying the function of calcineurin on the biosynthesis of GA can help us better understanding the mechanism of biosynthesis and regulation of GAs.