SOD And CAT On The Effects Of Antioxidative Responses Under Heat Stress In Aspergillus Niger 3.316

Aspergillus niger is one of the most widely used microbial strains in the enzyme fermentation industry.It can produce abundant enzymes and is an important cell factory.The accumulation of bio-heat formed in the fermentation process causes a high temperature environment to induce the formation of reactive oxygen radicals(ROS)in microbial cells and reduce the growth and reproduction speed of microbial cells and the activity of intracellular metabolic enzymes.As the main antioxidant enzymes,superoxide dismutase(SOD)and catalase(CAT)are important barriers against ROS damage.Therefore,study the characteristics of SOD and CAT on defense against cell damage and high temperature resistance,and explore Aspergillus niger The response mechanism of high temperature stress will lay a theoretical foundation for the breeding of new heat-resistant varieties of Aspergillus niger,which has important scientific significance and production value.In this study,the research object of Aspergillus niger 3.316 was used to explore the changes of Aspergillus niger temperature stress tolerance and cell morphology and structure under heat stress,and the changes in the activity of main antioxidant enzymes in the bacteria to the high temperature response antioxidant pathway influences.The main findings are as follows:1.Aspergillus niger 3.316 temperature stress tolerance and cell structure observation under heat stressThe temperature resistance test was carried out by the dry weight method of mycelium ball,and the optimum growth temperature of Aspergillus niger 3.316 was35?,the highest growth temperature was 47?,and the limit growth temperature was65 ?,40 min or 70 ?,20 min.Scanning electron microscopy and transmission electron microscopy were used to observe the changes of strains under the optimum growth temperature and stress growth temperature.It was found that the bacterial cells appeared rough,shriveled,thickened cell walls,and enlarged mitochondria.2.Aspergillus niger 3.316 whole genome sequencing and analysisThe Aspergillus niger 3.316 genomic DNA was extracted by the SDS method,and the whole genome was sequenced using the Pac Bio sequencing platform.After assembly and annotation,the genome was composed of 15 Contigs,with a total length of 34956132 bp and a GC% content of 49.21%,which is predicted 10032 coding genes,9998,6901,2118,9494 and 494 genes were annotated in the Nr,GO,KOG,KEGG,and CAZy databases respectively.Through the annotation of antioxidant genes,it was found that the main antioxidant genes were SOD and CAT.3.Aspergillus niger 3.316 SOD,CAT gene Cas9 knockout vector constructionThe SOD and CAT gene sequences in Aspergillus niger 3.316 were obtained by PCR amplification,and the vector required for constructing g RNA expression was obtained by the method of sequence synthesis.The SOD/CAT-g RNA gene fragment was obtained by PCR technology,and the gene fragment was obtained by two-step digestion and enzyme connection.Obtain the SOD and CAT gene Cas9 knockout vector p FC322-SOD/CAT-g RNA,and identify it by PCR and sequencing.4.Aspergillus niger 3.316 knockout vector transformation,identification and temperature tolerance test of knockout strainThe knockout vector was transformed into Aspergillus niger 3.316 germinating spore competent cells by electroporation to obtain the knockout strain,which was identified by PCR and sequencing,and the CAT knockout strain was obtained by temperature tolerance test on the knockout strain The optimum growth temperature is33?,the highest growth temperature is 45?,and the limit growth temperature is55?,40 min or 60?,30 min.The optimum growth temperature of SOD knockout strain is 30 ?,the highest growth temperature is 43 ?,and the limit growth temperature is 55?,40 min or 60?,30 min.