Construction Of Aspergillus Clavatus Transformants Library And Cloning And Functionl Analysis Of FlbA Gene Related To Conidiation

Aspergillus clavatus,a small filamentous fungus of the Aspergillus genus,is a common soil fungus that is widely distributed in warmer soils and in some foods.Aspergillus clavatus is actually not common in indoor environments,however,it is often associated with the brewing industry.It can be cultured on CZA,PDA,etc.The metabolism of Aspergillus clavatus can produce kinds of bioactive substances.There are abundant intracellular and extracellular hydrolase activity.Its secondary metabolites can inhibit certain fungal bacteria,such as patulin.Patulin,also known as protromycin,has a broad spectrum of antibiotic characteristics.At present,most of the research on Aspergillus clavatus,is related to its metabolism.However,there are few studies on sporulation related genes.In this study,strain Ac-32 from Aspergillus clavatus stored in this experiment was used as the research material.The main research and results are shown as follows.First,clone and analyze the related gene flbA of conidia formation of Aspergillus clavatus.According to the flbA gene sequence provided in NCBI database(www.ncbi.nlm.nih.gov),primers were designed with Primer 5.0 software.Then,PCR amplification was performed,the PCR products were gel electrophoresis and the target bands were cut and recovered.The DNA fragment was sequenced,and the tertiary structure and structural information of the coding protein were analyzed.The length of the gene was 2,208 bp.The nucleotide sequence of the gene was 69.9%similar to that of Aspergillus nidulans and 68.1% similar to that of Aspergillus niger.The homologous sequences of Aspergillus clavatus,Aspergillus nidulans and Aspergillus niger were found to be low.Aspergillus clavatus flbA gene encodes 734 amino acids.Its amino acid sequence homology with Aspergillus nidulans flbA was76.6%,and that with Aspergillus niger flbA was 81.6%Secondly,the construction of Agrobacterium tumefaciens-mediated transformation system of Aspergillus clavatus.The strains and plasmids used were Aspergillus clavatus Ac-32,Agrobacterium AGL-1 and plasmid pKD1.After co-culture,the transformant were screened by hypopycin B resistant plate,and then PCR and Southern hybridization were verified and analyzed.Finally,optimize the factors that affect the conversion efficiency.Different conditions in the conversion process have a great impact on the conversion efficiency.The optimum co-culture time and temperature were 2 d and 32oC,respectively.When the absorption value at the wavelength of 600 nm is 0.6,it is the optimal concentration of Agrobacterium,and the concentration of AS is 300 mol/L.These optimization experiments laid the foundation for subsequent gene knockout.Thirdly,the gene knockout vector of Aspergillus clavatus flbA was constructed.The 5' and 3' ends of the flbA gene and the hph gene were amplified.Double-joint PCR was used to connect the 5'-end flanking sequence of flbA gene,the hph gene and the 3'-end flanking sequence of flbA gene.Finally,T4 DNA ligase was used to connect the linear pKO1 B plasmid and flbA gene knockout box obtained after double enzyme digestion.The gene knockout vector of Aspergillus clavatus flbA was transformed into Agrobacterium tumefaciens.Fourthly,the gene knockout of Aspergillus clavatus flbA and the growth and metabolism analysis of gene deletion mutant.By means of Agrobacterium-mediated method,the knockout vector was transformed into the cells of Aspergillus clavatus,and a knockout strain of Aspergillus clavatus flbA gene was successfully obtained.The colony morphology and microstructure were analyzed,and the changes of conidia formation,growth and metabolism of Aspergillus clavatus were analyzed.It was found that flbA gene regulated conidium formation,and conidia could not form normally after flbA gene deletion.And it also has some effect on the growth and secondary metabolism of Aspergillus clavatus.The growth rate of flbA gene deletion mutant was slowed down on PDA medium.The activity of hydrolase was also changed,and the activities of protease and starch of Aspergillus clavatus flbA gene deletion mutant were increased.According to HPLC analysis,compared with the wild type Aspergillus clavatus,the yield of the mutant Aspergillus clavatus flbA gene lovastatin increased by 25.3%,and the yield of patulin was reduced by 7.8%.Therefore,flbA gene also has a certain effect on the growth and secondary metabolism of Aspergillus clavatus.