Construction Of PgxB Gene Disruptant In Aspergillus Niger And Characteristic Research

Aspergillus niger,a saprophytic fungus in nature,is widely distributed in soil,air and cereals and can cause mildew and rot in food,cereal,fruit and vegetables.It is a typical infectious fungi for juicy fruits such as apple,pear,peach and pomegranate.The main component of plant cell wall-pectin is polysaccharide chain that is formed by the linkages of different degree of esterification-galacturonic acid by ?-1,4-glycosidic bond.Pectinases are the main degrading enzymes for phytopathogen to breakthrough plant cell wall and polygalacturonase and polygalacturonase(PG)is one of the main members.As an industrial fermentative microorganism,A.niger is also used in the production of pectinase and applied in the food processing industry.Studies have shown that,for some fungi and plants,PG is virulence factor in fungi,but there is no report about pectinase gene in A.niger.Therefore,we studied the pathogenicity role of polygalacturonase gene pgxB in A.niger,which is important for the study of the function of pectinase gene in A.niger and the storage of postharvest fruits and vegetables.Gene knockout is the most direct way to study gene function.We used Aspergillus niger MA70.15(?kusA,pyrG-)as material,pyrG as selective mark gene(pyrG encodes orotidine-5-phosphate decarboxylase,cell lacking this enzyme cannot grow without exogenous uridine,but can resist toxicity of 5-Fluoroorotic acid)and deleted exo-polygalacturonase gene pgxB via homologous recombination.Firstly,We obtained the A.niger pgxB gene sequence from the Aspergillus genome database and amplified fragment AB(548bp)and CD(564bp)flanking pgxB and linked them together.Secondly,fragment ABCD was linked with plasmid pC3(5865bp,containing pyrG)to form knockout vector p C3-ABCD(6959bp)and it was transformed into A.niger MA70.15 protoplast via PEG transformation method.Lastly,?pgxB mutant was obtained by bidirectional screening of pyrG and confirmation.It was found that ?pgxB mutant showed lower pectinase activity(5.8%)compared with wild type and less virulent on apple and pear fruits as the necrosis diameter caused by ?pgxB mutant was significantly smaller(20%)than that of wild type.Further experiments showed that,in the process of infection in apple fruits,gene expression of polygalacturonase genes pgaI,pgaII,pgaA,pgaC,pgaD and pgaE was enhanced in ?pgxB mutant in comparison to wild type and the possible cause is compensating for the absence of the pgxB function,but the virulence of ?pgxB mutant still decreased significantly.Above results indicated that pgxB is an important virulence factor in A.niger and play a role in the postharvest storage process of apple and pear.Further research indicated the absence of pgxB did not affect the colony growth of A.niger on PDA and pectin media.We also found that the wild type and ?pgxB mutant had no significant difference in sensitivity to heavy metal ions stress such as aluminum,zinc,cadmium and copper,specifically manifested in the colony diameter and growth amount,indicating that the polygalacturonase gene pgxB had no correlation with the sensitivity of A.niger to heavy metal stress.However,heavy metals can decrease pectinase production by A.niger.Studies on exogenous influencing factors of pectinase production showed that pectin can induce the synthesis of pectinase and significantly increase the expression of pectinase genes,while glucose and D-galacturonic acid(0.1% and 1%)inhibited the synthesis of pectinase.Weak acid condition was most suitable for A.niger to produce pectinase,but 5% citric acid was most effective in inhibiting pectinase production.Through the above studies,we get the conclusion that pgxB is an important pathogenic factor in A.niger,which not only reduced the activity of pectinase,but also weakened the pathogenicity of A.niger to apple and pear,but it has nothing to do with the sensitivity of A.niger to heavy metals.