Study On The Effects Of Mu Ltiple Genes Knocked Out On Growth Aspergillus Niger

In this study,the binary vector which used hygromycin B-resistant and bleomycin as selection markers was introduced into Agrobacterium tumefaciens AGL1 by electroporation.The influence of different conditions on Agrobacterium mediated transformation efficiency were performed to improve the transformation efficiency as follows:A.tumefaciens was cultivated with 400 ?mol/L acetosyringone(AS)at 28? and 100 r/min for 5 h in 100 mL conical flask,co-cultivation at 1:100 ratio of conidia to bacterium was conducted on nitrocellulose membrane at 24? for 48 h.Based on the above optimized conditions,the transformation efficiency of A.niger achieved 35 transformants/107 conidia,an increase by 78%compared to that before optimization.Also,all the selected hygromycin B-resistant transformants were genetically stable after 15 generation subcultures on complete medium.The establishment of this transformation system can provide basics for transformation of filamentous fungi in the future.In the process of optimizing the transformation efficiency,obvious morphological changes were observed in some mutants and sequence analysis of T-DNA inserted site was performed in one conidiation-defective mutant.Two copies of hygromycin B-resistant genes were defected through Southern blot.The two integration sites of T-DNA gene into A.niger genome was determined by whole genome sequencing.The resulted showed that the T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination,which were two genes spt7 and beta-glucosidase D(bglD)mutational by sequence alignment analysis.By homologous recombination,the ?spt7,?brlA,?bglD single deleted mutant strains,were constructed based on the optimization of the efficiency of ATMT in A.niger.It turned out that the deletion of spt7 gene was the key factor of conidiation-defective in A.niger.The phenotype of ?spt7 mutant,its hyphae pinched off and inhibited the growth severely,was different from the phenotype of ?brlA mutant which was important in conidial central regulatory pathway.The distinct differences between them were the aerial hyphae.The hyphae of ?brlA mutant grew well,though the conidiophore failed to develop.In order to understand the influence of the deletion of spt7 gene in A.niger,transcriptome sequencing technology was used to sequence the transcriptome of ?spt7 single mutant.The research resulted that a total of 1,430 differentially expressed genes were detected between two samples,in which 1,036 genes were downregulated and 394 genes were upregulated.The deletion of spt7 gene had obvious inhibition on amino acid metabolism,amino sugar and nucleotide sugar metabolism,fatty acid metabolism,glycolysis/gluconeogenesis and metabolism.The distinct influence of this gene deletion was mitogen activated protein kinase by protein interactions analysis.Each mutant strains and original strain were cultured under different carbon sources and found the growth situations of various strains in different carbon sources were similar.It turned that spt7 gene deletion cannot be recovered through adding exogenous carbon source.By fermentation experiments of the mutants and original strain,the results showed that the citric acid production of the original strain and ?bglD mutant were 66.66g/L,66.45g/L respectively,but the citric acid production of ?spt7,AbrlA,53 mutant were 8.01 g/L,7.12 g/L,7.81 g/L respectively.The citric acid production was fell by 87.98%,89.32%,88.28%compared by the original strains.It showed that spt7 gene was the key gene in A.niger to affect its growth metabolism,rather than gene,and the formation of the spores was beneficial to the synthesis of the citric acid from A.niger.In the determination of energy,the contents of ATP,NADH in ?spt7,53 mutants were always less than the original strain,but the change trend was consistent with the original strain,and differentiated significantly,indicating that the deletion of spt7 gene in seriously affect energy metabolism of A.niger.The ATP,NADH variation ranges of ?brlA mutant was less than other mutant strains,but its content less than the original strain,indicating that the formation of conidia could promote energy metabolism in Aspergillus niger.