Study On Genes In The Nad Metabolic Pathways Related To Growth Of Fusarium Oxysporum

Fusarium spp.has a wide variety and distribution,and is commonly found in soil and found in animal and plant organisms,causing widespread infection in humans,animals,and plants.Clinically,Fusarium can cause superficial or deep infections,which are commonly caused by fungal keratitis and can cause blindness in severe cases;Deep infection is commonly seen in patients with low immune function,mainly manifested as invasive infection,with a high mortality rate.In agriculture,Fusarium is one of the most important plant pathogenic fungi,which can infect plants or crops and cause Fusarium wilt and root rot.Its toxic secondary metabolites can cause poisoning and damage to humans and animals.Fusarium exhibits a certain degree of resistance to commonly used clinical antifungal drugs,leading to poor therapeutic efficacy.Nicotinamide adenine dinucleotide(NAD),its reduced form(NADH)and phosphorylated form(NADP)are the core of cell metabolism and energy production.During aerobic respiration,NAD acts as an electron transmitter and shuttles through the oxidative respiratory chain,eventually synthesizing ATP,which provides energy for cells.Therefore,maintaining NAD concentrations is important for cell and biological viability.There are three main biosynthetic pathways of NAD,namely,the Preiss – Handler(PH)pathway,the de novo synthesis pathway,and the salvage pathway,which collectively regulate NAD homeostasis.NAD regulates a variety of cellular functions,such as plants,animals and microorganisms,and plays an important role in energy metabolism,cell apoptosis and aging.Currently,there are few reports on the role of NAD metabolism in the filamentous fungi.This study aimed to find genes in the NAD metabolic pathways associated with Fusarium oxysporum growth and drug sensitivity,providing potential targets or strategies for the control of Fusarium oxysporum.Through the analysis of the NAD metabolic pathways of Fusarium oxysporum,five important catalytic enzymes in these pathways were selected as targets,namely nicotinic acid phosphoribosyltransferase(Npt1),quinolinic acid phosphoribosyltransferase(Bna6),nicotinamide ribokinase(Nrk1),5 ’-nucleotidase(5’-nucleiotidase),and NAD kinase(NADK).Its coding gene is FOXG＿01191,FOXG＿06246,FOXG＿13827,FOXG＿04408,FOXG＿00298.The previous laboratory has established and successfully optimized an Agrobacterimu tumefaciens-mediated transformation system(ATMT)for Fusarium oxysporum.Using ATMT technology,five Gene mutants has been successfully constructed to analyze the function of the gene.One mutant strain with abnormal growth ΔNpt1 was obtained,and compared with the wild strain,ΔNpt1 hyphal growth retardation,pigment production was reduced,and the production of conidia is reduced,indicating that FOXG＿01191 gene was related to growth.The drug sensitivity of five mutants to four commonly used clinical antifungal drugs itraconazole,voriconazole,amphotericin B,and caspofungin was tested.The results showed that none of the five mutants had obvious changes,indicating that the five genes were not related to drug resistance.The NAD metabolic pathways of mutants ΔNpt1 was analyzed and the results showed that the NAD and ATP content decreased.The expression levels of NAD metabolism pathway related genes in ΔNpt1 were detected by q PCR,and the results showed that the expression levels of Nma and Qns1 were significantly down-regulated.The results show that Npt1 deletion leads to the obstruction of Preiss-Handler pathway,the decrease of NAD synthesis,the imbalance of NAD redox homeostasis,and the decrease of ATP release in the oxidative phosphorylation pathway.In summary,this study conducted analyzed the relationship between Npt1 and growth,and found that the gene encoding Npt1,FOXG＿01191,is an important gene in the regulation of NAD biosynthesis.It affects the synthesis of NAD through the regulation of Preiss-Handler pathway metabolism,and can regulate ATP synthesis,which affects the growth of Fusarium oxysporum.These results provided a theoretical basis for revealing the growth mechanism of pathogenic fungi and seeking potential therapeutic targets.