Functional Study And Application Of Aspergillus Niger Spt7 Gene

The SAGA complex is a multifunctional protein complex responsible for more than10% of intracellular gene transcription.spt7 serves as the core protein of the SAGA complex and maintains the stability of the SAGA complex.No Spt7-related studies have been reported in fungi other than yeast.Therefore,in this study,Spt7 of Aspergillus niger origin was knocked down and overexpressed to observe the characterization as well as the transcriptional level on Aspergillus niger.In this study,the amino acid sequences of Spt7 from different Aspergillus niger,Cladosporium mitis and other filamentous fungal sources were compared in multiple sequences and the protein structures were analyzed,and it was found that Spt7 was less consistent in different species,but all of them had conserved Bromo structural domains.Aspergillus niger 1062 was used as the starting strain,and the plasmid p CS,which overexpresses spt7,and the plasmid p C-S,which knockdown spt7,were transferred into the1062 strain by Agrobacterium transformation method to obtain Aspergillus niger OEspt7 transformant 1062-p CS and Aspergillus niger △spt7 transformant 1062-p C-S.The growth patterns of OEspt7 transformant,△spt7 transformant and Spt7 was found to be essential for mycelial growth,sporulation and spore maturation of the fungus.The number of spores per unit area of the overexpressed spt7 transformants was three times higher than that of the control group,while the number of spores was significantly reduced and almost uncountable after knocking out spt7,after the colony sporulation and late spore maturation.In addition,we observed the resistance of transformants and controls and found that either spt7 knockdown or overexpression of spt7 improved the antioxidant properties of A.niger.We hypothesized that overexpression of spt7 increased the transcript levels of antioxidant enzymes in the bacterium,so the SOD,GPX transcript levels in the bacterium increased 3.97-fold,4.11-fold and 0.76-fold,respectively,making the bacterium more resistant to H2O2.In contrast,knockdown of spt7 induced a splice cross-protection mechanism in the bacterium,leading to an increase in the level of Cpe B transcript in the bacterium,which was 1.76-fold higher than that in the control group,thus increasing the antioxidant resistance of the bacterium.However,in terms of heat resistance of the bacterium,the heat resistance of the overexpressing spt7 bacterium was improved.After heat stress induction by 42°C water bath,the heat stress protein and heat stress transcription factor were detected in the spt7 overexpressing transformants and the control group,in which the transcript levels of Hsp70,Hsp90 were increased 1.42-fold and 2.14-fold,respectively.The heat stress transcription factor Hsf was increased by 1.49-fold.However,the knockout strain was unable to grow and germinate at 42°C.So spt7 has a direct effect on the heat resistance of the bacterium.Based on the results of the study in strain 1062,we applied the aspect of spt7 to improve the heat resistance of the bacterium.The high citric acid producing strain TNA09 was used as the starting strain to overexpress spt7,and the transformants were subjected to high temperature shake flask fermentation.And microscopic observation of mycelium seeking morphology,the morphology of mycelium balls intact under high temperature,indicating that overexpression of spt7 can maintain the morphology of the bacterium under high temperature,and thus reduce the effect of high temperature on acid production.