Functional Analysis Of UDP-glucose Pyrophosphorylase And Phosphoglucomutase In The Carbon Metabolism Of Ganoderma Lucidum

Ganoderma lucidum is a well-known large medicinal fungi in Asia,because of its high nutritional and medicinal value,and it has a long history of application in these countries.The polysaccharide is one of the main medicinal ingredients of G.lucidum,it has many functions such as antineoplastic,hypoglycemic,hypolipidemic and hepatoprotective effect.The production of polysaccharide is one of the important index to measure the quality of G lucidum,recently,the research on polysaccharides of G.lucidum were mainly focus on how to improve the yield.Researches on polysaccharide biosynthesis were mainly concentrated in the fermentation parameters and exogenous addition of inducer to improve the yield of polysaccharide.The mechanism of polysaccharide synthesis was preliminarily predicted by detecting the activity of key enzymes in polysaccharide biosynthetic pathway in G lucidum.Presently,the genome sequencing of G lucidum provided data for us to research the mechanism of the polysaccharide biosynthesis at the level of molecular genetics,and these promoted the research progress of polysaccharide synthese in G.lucidum,in turn increased the value of scientific research and commercial application.In this paper,we first systematic research the functions of two key enzymes,UDP-glucose pyrophosphorylase(UGPase)and phosphoglucomutase(PGM),in polysaccharide biosynthesis pathway of G lucidum.We also studied the role of the UGPase and PGM in the progress of the physiology and metabolism in G lucidum.The main researches as following:1.The function analysis of UGPaseUGPase occupies a very important position in the carbon metabolism pathway,at present,studies on UGPase were mainly concentrated in animals,plants and bacteria,and it had found that the UGPase has participated in many biological effects.However,the study on the function of UGPase in fungi especially on macro fungi is still very little.In this research,basis on successfully sequenced G lucidum genome sequences,we have obtained the sequences which encoding UGPase and PGM from the genome of G lucidum.Then the bioinformatic analysis showed that UGPase has the functional domain of UDPGP conservative.Then,we amplified the sequences separately from the predict gDNA and cDNA,then the analysis between the two sequences showed that it has 8 typical fungal introns.We obtained the recombinant protein UGPase by prokaryotic expression method,and analysis on the activity of this enzyme showed that it can catalyze glucose-1-phosphate(Glc-l-P)and UDP-glucose(UDP-Glc)in vitro,and the Km values were 31.03 ?M and 773.8 ?M,and the kcat values were 33.11 s-1 and 0.211 s-1 separately aganst to the substrate of Glc-1-P and UDP-Glc.According to this,we identified this protein is a functional protein,and the reaction catalyzed by the recombinant protein was freely reversible in vitro.Then,we analyzed the function of the UGPase by silencing its encoding gene,and we found that the contents of the two key metabolic intermediates that genes control were changed in vivo of G.lucidum,and the gene knockdown can affect the polysaccharide biosynthesis and cell wall components and structure of G.lucidum.The intracellular Glc-1-P was accumulated while the UDP-Glc content was reduced in the ugp silencing strains(ugpi),further explained in the direction of UGPase catalysis was inclined to the UDP-Glc biosynthesis in vivo;secondly,ugpi strains also showed the biosynthesis of the extrapolysaccharide was decreased in G lucidum,the content of the main component of cell wall,glucan,was also reduced,and the cell wall structure was changed.2.Study on the regulation mechanism of ugp.on the hyphal branching in G.lucidum:In our study,we didn't only found the functions of ugp which were the same with the other species,but we also found that the expression of ugp can affect the number of hyphal branching in G.lucidum.In order to confirm that ugp has regulatory effect on hyphal branching in G.lucidum,therefore,we constructed the ugp over-expression transformants.The results showed that the ugp over-expression transformants indeed influenced hyphal branching of G lucidum,and the cytosolic calcium content was also changed in the ugp over-expression transformants.In order to verify whether the effect of ugp on hyphal branching was through regulating the level of the cytosolic calcium,we used the exogenous calcium and calcium chelate to detect the hyphal branches.And the results showed that the increased number of hyphal branches in ugpi strains was rescoved by adding the calcium,and the decreased number of hyphal branches was also rescoved by adding EGTA.Further study found that the change of cytosolic calcium was regulated by the level of the intracellular Glc-1-P,and the exogenous Glc-1-P addition has also affected the cytosolic calcium level and the number of the hyphal branches in G lucidum.These results further suggested that the hyphal branching,which was influenced by the expression of the ugp,was regulated by the intracellular metabolic intermediates Glc-1-P in G lucidum.3.The function analysis of PGM:PGM the first enzyme in the pathway of nucleotide sugars synthesis,and it could affect the carbon flow in carbon metabolism,so it has important physiological functions in almost species.At present,there are some researches on the function of pgm in animals,plants and fungi,but the study on large basidiomycetes is deficient.In this research,we obtained the pgm gene through homologous comparison,and through using RNA silencing to study its function in carbon metabolism.Results showed that silencing leads to the change of the flow metabolism of polysaccharide synthesis,the extracellular polysaccharide content was significant decreased,and the concentration of cytosolic calcium was significantly increased with the reduced number of hyphal branches.And the level of the intracellular ROS was also increased and the number of the mitochondrion was also increased in the pgmi strains.In sum,the function of the key enzymes,UGPase and PGM,in carbon metabolism pathway was studied by using RNA silencing and overexpression,and abnormal carbon metabolic will cause changes in calcium signal.These results provide a favorable opportunity for potential mechanism between the carbon signaling and calcium signaling in fungi,and indicate that the possibility of this interaction signal mechanism may also exist in plants and microorganisms.