Construction Of Heterologous Expression Fusarium Strains And Virulence Analysis Of Gly Mutant Strains

Fusarium expression systems having a high potential for eukaryotic protain expression, fungal expression systems have many advantages, such as high extracellular secretion, large expression capacity, capable of post-translational protein modifications, for exmpale, the glycosylation of protein, the exogenous proteins having natural activity, Fusarium expression systems have easy storage features because of Fusarium easy to generate hyphae and spores.Phytic acid (myo-inositol hexakisphosphate, IP6) is the main stored forms of phosphorus in plant. In vivo, monogastric animals (such as chickens, cattle, pigs, etc.) can not effectively utilze the feed rich in phytic acid for the lacking of the degradation enzymes. Thus adding phatase to the feed has many advantages, for instance, improving the monogastric animals’utilization of phosphorus in plant feed, strengthening the absorption of mineral elements, reducing the phosphorus content in animal waste, and lowering phosphorus pollution to the environment et al. Therefore, application prospect of phytase as a feed additive is very broad.Antimicrobial peptides (AMPs), widely present in a variety of organisms, is a class of small peptides. When organisms is attacked by pathogenic microorganisms or stimulated by external factors, AMPs is rapidly generated. AMPs has a variety of biological activity, including a broad-spectrum bactericidal effect on pathogenic microorganisms such as bacteria, fungi, viruses and protozoa, regulating the immune function, inhibiting and killing cancerous cells. With safe, broad-spectrum, non-resistance and other biological characteristics, AMPs has been developed into a new type of antibiotic alternative to traditional antibiotics and has broad development in the fields of medicine, agriculture and food as well.In this study, based on the published sequence of bacterial phytase gene (APPA) and the sequence of defensin separation from saprophytic ascomycetes (Pseudoplectania nigrella), adding a signal peptide derived from yeast SUC2 gene encoding a transferase enzyme before the heterologous proteins coding sequence, making the expression of heterologous proteins can be secreted into the extracellular, and using the constitutive promoter of three glyceraldehyde phosphate so that the heterologous proteins can be efficiently express, we constructed two vectors:one containing a exogenous gene of phytase and the homologous regions of the Tri5 gene which is the key gene in producing the toxin of deoxynivalenol (DON) and the other containing a exogenous gene of defensin and the homologous regions of the Pks4 gene which is the key gene in producing the toxin of zearalenone(ZON). Using PEG-mediated protoplast transformation method, we transformed the Fusarium graminearum 5035 which is a predominant strain causing Fusarium Head Blight. The transformants were identified with PCR and Southern blot. Finally, mutants were determined by enzyme activity analysis.In this study, we constructed Gly deletion mutants of different strains of Fusarium graminearum, and analysis mutants’ virulence and toxin production capacity. We found \Gly deletion strains compared to wild-type, toxin production and virulence has reduced both.