Preliminary Study On Function Of Ras Gene From Aspergillus Niger

Aspergillus niger is a common species in the industrial production of Aspergillus,which is widely used because of its large amount of metabolites and rich enzyme system.The advent of the post-genome era has also pushed the research of Aspergillus niger into the field of genetic engineering.As an important member of the small G protein,the research of the multi-functional cytokine Ras has been increasing,but there are still not many reports about Ras in the fungus.The function of the ras gene in Aspergillus niger is still unclear.Therefore,use the method of molecular biology to explore the function of the ras genes in Aspergillus niger,on the one hand to lay the foundation for the transformation of strains,on the other hand will also play a role in the in-depth study of small G protein in the fungus..This article uses gene overexpression to increase the expression of ras gene.The effect of ras gene on the growth and metabolism of Aspergillus Niger CGMCC 3.316 was preliminarily investigated by analyzing the differences in the biological characteristics of transmutants and wild-type strains.First,the coding sequence of the ras gene was predicted using the partially conserved sequence of the ras gene known in the laboratory and two sequences with higher homology in the NCBI.Then,the appropriate gene restriction sites were added to both sides of the target gene,and were digested,ligated and transformed with pCAMBIA 1301 plasmid and pBARGPE1-Hygro-EGFP plasmid to complete the construction of two types of overexpression plasmids.Next,the PEG-mediated protoplast transformation method was used to transform the plasmid,screening with hygromycin B resistance transformation,and some of the resistant transformants were selected for genetic stability of the target gene.A total of 8 strains with both hygromycin B resistance and genetically stable transformants were obtained,namely as 2 ck pCAMBIA 1301 empty plasmid transformation,2 pBARGPE1 empty plasmid transformation,2 overexpression transformant pCAMBIA1301:ras(pC:ras)and 2 overexpression transformant pBARGPE1:ras(pB:ras).Finally,biological differences between transformants and wild type were tested.(1)RT-PCR results showed that the expression of ras gene in the two transformants of pC:ras and pB:ras increased 4.49 times and 7.82 times,respectively.Western results showed that Ras protein expression also increased.(2)By measuring colony radius,it was found that the growth rate of pC:ras and pB:ras converters was 1.10 times and 1.17 times respectively than that of wild type strains,and the biomass was 1.53 times and1.98 times respectively.(3)By liquid fermentation test,the cellulase activity of pC:rasand pB:ras was 1.30 times and 1.44 times higher than that of the wild type,and the citric acid was 1.40 times and 1.86 times respectively.The pB:ras transformants were superior to the pC:ras transformants both in terms of transformation efficiency and overexpression.The results of this experiment indicate initially that the overexpression of ras gene of Aspergillus niger promotes the growth and metabolism of Aspergillus niger.