Effects Of Laea Gene Silencing And Overexpression On Metabolism Of Ganoderma Lucidum

Throughout the whole process of Ganoderma lucidum and its research status,the low yield of active substances is the main reason why fermentation can not be commercialized.Studies have shown that by constructing high expression engineering strains,the biosynthesis amount of ganoderic acid can be significantly improved.Therefore,transgenic technology is an effective strategy for increasing the production of active substances.Most of the current research focuses on over-expression of key enzyme genes in the biosynthesis pathway of ganoderic acid or ganoderma lucidum polysaccharides.LaeA is one of the global regulators of filamentous fungi and is involved in the regulation of morphological and secondary metabolism.Studies showed that LaeA gene had a positive regulatory effect on the expression level of secondary metabolism-related gene clusters and the production of secondary metabolites.Ganoderma lucidum,as one of the higher filamentous fungi,has not been reported whether its morphological and secondary metabolism is regulated by LaeA gene,how it regulates and its internal regulatory mechanism.The transgenic technology was used to construct the LaeA gene silencing and overexpressing strains to explore the effects of LaeA gene on morphological and secondary metabolism of Ganoderma lucidum and its intrinsic regulation mechanism.By studying the effects of LaeA gene silencing,overexpression of LaeA gene cDNA sequence and overexpression of LaeA gene sequence on morphological development and secondary metabolism of G.lucidum,the results showed that:(1)LaeA gene silencing strains grow slowly or even stagnant,mycelial morphology changes,bifurcation increases,mycelial volume decreases,sporulation ability decreases;biomass decreases,sugar consumption decreases;total ganoderic acid and polysaccharides were reduced;the expression levels of hmgr,sqs,ls and ugp,pgm,gls were in dynamic equilibrium.(2)The mycelial growth of the overexpressed LaeA gene cDNA sequence was significantly accelerated.The hyphae radius was 4.5 times of the WT strain on the third day.The mycelium was thick and tough,the morphology was changed,the bifurcation was increased,but the sporulation ability was lost.Cell biomass decreased significantly,but sugar consumption was significantly up-regulated;Ganoderic acid was not produced,polysaccharide synthesis was down-regulated;the expression levels of hmgr,sqs,ls and ugp,pgm,gls were significantly reduced or even not expressed.(3)LaeA gene overexpressing strain showed significantly increased mycelial growth.The mycelial radius was 4.0 times of WT strain on the third day of culture,and the sporulation ability was significantly enhanced,but the mycelial structure did not change;the biomass increased significantly,and the sugar consumption accelerated;the production of ganoderic acid,lanosterol,intracellular polysaccharide and extracellular polysaccharide were up-regulated,the maximum amount were 3.62,3.58,1.42 and 1.78 times of WT strain,respectively;the expression levels of hmgr,sqs,ls were up-regulated.The maximum expression level was 23.74,2.12,and 8.62 times of WT strain,respectively.Polysaccharide-associated gene ugp was up-regulated on day 3 of culture and was 1.65 times higher than WT strain,but ugp gene expression was significantly down-regulated during subsequent culture.The expression levels of pgm and gls were up-regulated,and the maximum expression was 10.73 and 3.38 times of WT strain,respectively.In summary,this study explored the regulation of LaeA gene on morphological and secondary metabolism of Ganoderma lucidum for the first time.This study found that LaeA gene was involved in the regulation of morphological and secondary metabolism of Ganoderma lucidum,and had a positive regulatory effect on the expression level and yield of ganoderic acid and polysaccharide-related gene clusters.This study indicates that the construction of LaeA gene high expression engineering strains by transgenic technology is one of the strategies for increasing the production of Ganoderma lucidum.At the same time,this study provides theoretical support for further elucidation of the regulation mechanism of LaeA gene on the metabolism of filamentous fungi.