The Construction And Function Of The Gene SodC Deletion Mutant

Aspergillus niger is one of the most common species found in fungal communities.It is an important fermentation industrial strain,and is also known to cause the most severe symptoms in fruit during long term storage.Superoxide dismutases?SOD,EC 1.15.1.1?are metalloenzymes that form a primary cellular antioxidant defense system by catalyzing the superoxide disproportionation reaction to produce molecular oxygen.It is composed of two subunits,and each subunit contains one Cu2+ and one Zn2+.We try to investigate the role in terms of physiological metabolis of the gene sodC in A.niger.sodC gene was deleted in A.niger by the principal of homologous recombination.Firstly,the upstream and downstream flanking fragments of the sodC gene were amplified by using the genomic DNA of Aspergillus niger as template.Then the upstream and downstream flanking fragments were connected with PCR by using overlapping sequences.The fragment ABCD was connected with plasmid pC3 and transferred to DH5? competent cells.The recombinant plasmid pC3-ABCD was constructed successfully by blue spot screening and PCR identification.The recombinant plasmid was transferred into protoplast of Aspergillus niger by CaCl₂/PEG transformation method.Through multi-step screening and PCR identification,we can get the deletion mutant ?sod C.In order to the further research,the wild type A.niger and ?sodC mutant are as experimental materials.By comparing the growth and colony diameter of wild type A.niger and ?sodC mutant in normal condition,it can be found that there is no difference between the wild type A.niger and ?sodC mutant.All of these suggest that sodC is not required for the growth of A.niger.The deletion of sodC leading to the decrease of whole SOD activity in A.niger suggests that sodC did contribute to full enzyme activity.Therefore,we observe the mycelium growth and spore germination of wild type A.niger and ?sodC mutant in these stress conditions of high concentrations of metal ions?such as Cu2+,Zn2+,Al3+,Cd2+?,H₂O₂ and menadione?a generator of intracellular ·O2–?,which can induce oxidative stress to organisms.Menadione could significantly inhibit the mycelium growth of the ?sodC.Further results showed that spore germination of the ?sodC was significantly inhibited by menadione compared with that of wild type.At the same time,the contents of ROS and MDA in mutant are higher than that of wild type under the condition of menadione.When the wild type A.niger and ?sodC mutant pretreated with ascorbic acid?a natural antioxidant?,we found that the mycelia of the wild type A.niger grew faster than the mycelia treated directly with the menadione.These results suggest that sodC gene of A.niger plays an important role in the antioxidant activity in the process of resisting oxidative stress.In addition,with the deepening of the damage,the sodC deletion mutant can significantly delay the infection of fruits compared with the wild type.The contents of ROS and MDA in the fruits were detected,and the contents of ROS and MDA in the fruits infected by ?sodC mutant were significantly lower than that of the wild type.The RNA of the wild type A.niger and ?sodC mutant infecting fruits were extracted.The expressions of the SOD-encoding genes?sodB,An04g04870,An08g03890,An16g05520?of the ?sodC mutant were significantly higher than that of the wild type in the infection condition.The results showed that sodC deletion mutant caused reduced virulence on fruit,which further support the hypothesis that SOD is important for the full virulence of A.niger.All of these proved that sodC plays an important role in the antioxidant activity in Aspergillus niger,and it is an important gene involved in fruit infection.Catalase?CAT,EC 1.11.1.6.?is an important member in the antioxidant enzyme system of A.niger.We explore the role in the process of metabolism in A.niger by comparing the phenotype between the ?cat A mutant and the wild type strain.The results showed that there was no difference between the two groups under normal culture conditions in the mycelia growth and the colony diameter.The results showed that the catA gene plays no an important role on the growth of A.niger under normal culture conditions.The activity of CAT was detected in both strains,and it was found that the deletion of catA gene resulted in the decrease of the whole level of catalase in A.niger.It was also found that H₂O₂ had a significant inhibitory effect on the mycelia growth.At the same time,compared the infection ability to the fruits,the lesion diameter caused by ?catA was smaller than that of the wild type in A.niger.These results have proved that the catA gene may play an important role in the antioxidant activity of the growth in A.niger.