Functional Analysis Of 4-coumarate: CoA Ligase Gene (4-CL) In Ganoderma Lucidum

Ganoderma lucidum,as a large fungus with homology of medicine and food,is not only a traditional Chinese medicine but also a health food rich in various active substances.Polysaccharides,ganoderic acids,steroids and flavonoids in G.lucidum are the main active ingredients,which have anti-cancer,cholesterol-lowering,liver-protecting and other pharmacological effects.Studies have shown that ethylene can effectively improve the secondary metabolites in G.lucidum,such as ganoderic acids,flavonoids,etc.,but its regulatory mechanism is still unclear,which restricts the medicinal development of G.lucidum.In this study,through the analysis of transcriptome and metabolome after ethylene treatment of G.lucidum,three differentially expressed genes related to the synthesis of secondary metabolites of G.lucidum were screened and obtained,namely terpenoid synthase gene(GL24515),β-glucosidase gene(GL27550)and 4-coumarate coenzyme A ligase gene(GL21040).Among them,4-coumarate coenzyme A ligase(4-CL)is a key enzyme in flavonoid biosynthesis and lignin synthesis pathway,which not only regulates the biosynthesis of secondary metabolites.It also regulates the growth and development of plant.Therefore,in order to further analyze the biological function of 4-CL in G.lucidum,RNAi and overexpression vectors of 4-CL gene were constructed in this study.The main results obtained in this study:1.Two interference strains(GL21040i-19,GL21040i-43)were obtained by q RT-PCR screening,and their GL21040 gene expression levels were 22.02% and 17.60% of the expression level of wild type strain(WT),respectively.The expression levels of GL21040 gene in two overexpression strains(OE: GL21040-47,OE: GL21040-54)were 19.18 times and 15.65 times that of wild-type strains.2.By measuring the flavonoid contents of interference and overexpression strains,it was found that the flavonoid contents of interference strains(GL21040i-19,GL21040i-43)were2.91% and 2.52%,respectively.Compared with the flavonoid contents of wild-type strain WT(3.25%),the flavonoid contents of interference strains decreased by 10.46% and 22.46%,respectively.The flavonoid contents of overexpression strains(OE: GL21040-47,OE:GL21040-54)were 4.45% and 3.52%,respectively.Compared with WT,the flavonoid contents of overexpression strains increased by 36.92% and 8.31%,respectively.The results showed that 4-CL(GL21040)had a regulatory effect on the biosynthesis of flavonoids in G.lucidum.3.By analyzing the ultrastructure of the cell wall of the 4-CL transformants,the results showed that the cell wall of the GL21040 interference strains were looser,less dense,and the boundary was not clear compared with the cell wall of the wild-type strain,while the cell wall of the GL21040 overexpression strains were tighter,dense,and the boundary was more obvious compared with the cell wall of the wild-type strain.4.Through the fruiting experiment of G.lucidum,it was found that the primordium formation time of GL21040 interference strains were 10 days later than that of wild type strain,while the primordium formation time of overexpression strains were 3 days earlier than that of wild type strain.The results showed that 4-CL could regulate the primordium formation time in G.lucidum.5.By measuring the lignin content of G.lucidum fruiting bodies,it was found that the lignin contents of GL21040 interference strains(GL21040i-19,GL21040i-43)were 111.88mg/g and 89.25 mg/g,respectively.Compared with the lignin content of wild-type strain WT(139.17 mg/g),the lignin contents of GL21040 interference strains decreased by 19.61% and35.86%,respectively;the lignin contents of GL21040 overexpression strains(OE: GL21040-47,OE: GL21040-54)were 162.98 mg/g and 159.68 mg/g,respectively.Compared with wildtype strains,the lignin contents of GL21040 overexpression strains increased by 17.11% and14.74%,respectively.The results showed that 4-CL could regulate the synthesis of lignin in G.lucidum.In summary,the study found that G.lucidum 4-CL gene had a positive regulatory effect on the biosynthesis of flavonoids and lignin in G.lucidum,and participates in the cell wall synthesis,growth and development of fruiting bodies.These results will provide a preliminary basis for further study on the regulation mechanism of secondary metabolite biosynthesis in G.lucidum,and provide a theoretical basis for the genetic improvement of G.lucidum strains.