Silencing Of Ras In Aspergillus Niger And Primary Studies Of Its Effect On Mycelial Growth

Aspergillus niger is the most common fungus among filamentous fungi.It has been widely used in industrial production because of its excellent acid production.The ras gene belongs to the family of Ras of the small G protein family and plays an important role in the GTPase protein signaling pathway.The Ras family is important for cell proliferation,differentiation,apoptosis and gene regulation.At present,the ras gene of fungi is less reported,and it's not clear about the function of ras gene in Aspergillus niger.Therefore,it is important to analyze the function of ras gene in Aspergillus niger by means of molecular biology,which is of great significance to explore how the key genes of GTPase protein pathway regulate fungus growth and its metabolism.In this study,RNA interference(RNAi)technique was used to silence the ras gene in Aspergillus niger 3.316,and the silencing efficiency of the transformant was analyzed.Firstly,the conserved regions of ras gene were predicted by software,and primers were designed according to conserved regions.Two pairs of primers were designed with Aspergillus niger CBS513.88 as the template,and Lras and Sras gene fragments with length of 629 bp and 379 bp were cloned with Aspergillus niger 3.316 as template.And the pSilent-1 plasmids were successfully constructed by two-step double-cleavage.Two plasmids of S-pS-ras and L-pS-ras were successfully constructed.After the experiment,the optimum conditions for the preparation of Aspergillus niger protoplasts were obtained,and the protoplasts suitable for transformation were successfully prepared.Nine resistant strains were obtained by screening with hygromycin B resistant plate by PEG-mediated protoplast transformation and electroporation transformation.Four transformants were obtained after the target gene detection.Finally,the properties of the transformants were studied.(1)The silencing efficiency of the transformants was analyzed by RT-PCR.The results showed that the two transformants ras were down-regulated by 67.4% and 64.7%,respectively.(2)The mycelial growth rate of the transformants was measured,the sporulation conditions were observed and the biomass was determined.The growth rate of wild-type hyphae was 1.23 and 1.17 times of that of the two transformants respectively.When cultured under the same condition,the wild type can produce spores much earlier than the transformants.Microscopic observation of transformants and the wild type showed that the transformants produce thinner mycelial and the number of branches was significantly reduced.Plus,the biomass of wild type were 1.88 and 1.82 times of the two transformants.(3)The yield of transformants' ?-glucosidase,endoglucanase(Cx)and total acid were determined by liquid fermentation experiments.The results of this study indicate that ras gene may regulate the downstream genes of signal pathway and regulate the secretion of cellulase and acid.The silencing of this gene can inhibit the growth and metabolism of Aspergillus niger.