Construction And Biological Characteristics Of Ras Gene Knockout Promoter In Aspergillus Niger

Aspergillus niger, a saprophytic filamentous fungus,which widely exists in the environment, is now considered as a important strain in industrial production of enzyme and acid, especially in cellulase and organic acid producing. ras gene belongs to a Ras family of small G-protein, it mediates the GTPase protein s ignaling pathway which is the key signal transduction mechanisms for filamentous fungus regulating growth and gene expression after the recept of external environment signal. However, the function of ras gene in Aspergillus niger is still not understood so far, so the study of related genes in GTPase protein signaling pathway for growth and obtaining fungal metabolites has great significance.This study aimed to preliminary analyze the role of ras gene in Aspergillus niger 3.316 growth process through homologous recombination and research on the biological characteristics of the transformants. A pair of primers is designed based on ras gene and its upstream and downstream region sequence in the known genome of Aspergillus niger CBS513.88, then large fragment of ras gene was amplified by PCR with Aspergillus niger 3.316 as a template, sequenced for precise sequence information and we analyzed conserved regions of ras gene through bioinformatics software to identify homology arm length. The fragment is coupled with appropriate restriction sites and linked to the vector for the construction of knock-out plasmid, and we used PCR and digestion to verify the plasmid is successfully established. The knock-out plasmid is shifted to agrobacterium tumefaciens by freeze-thaw method, and we co-cultured transformants of agrobacterium tumefaciens and conidium of Aspergillus niger in IM induction medium to get the plasmid into the cell of Aspergillus niger and recombined in the genome. After preliminary screening in the plate with hygromycin( 250 μg/mL), we acquired 68 transformants and then screen by amplifying ras gene and hygromycin gene for the strain which is recombined to the correct position, and finally we obtained one ras gene knock out strain.What’s more, we studied biological characteristics of ras gene knock out strain. The result showed that the growth rate of wild type strain is approximately 2.18 times higer than knock-out strain. We also observed that mycelia of knock out strain grew compactly and got darker, and its mycelial growth and spore production occurred simultaneously, on the contrary, mycelia of the wild type strain grew crumbly and its spore production is behind mycelial growth. The biomass of wild type strain is 1.78 times higher than knock out strain. And we find that the change of temperature had no impact on knock out strain. Endoglycosidase and β-glucanase activity have been reduced in knock out strain. It showed that the knock out of the ras gene of Aspergillus niger could affect the growth, fission and spore production of mycelium and the asexual reproduction, and the cellulase activity was inhibited.