Functional Study Of The Sporulation-related Genes FlbD And StuA In Aspergillus Coronatus

Aspergillus cristatus,the key fungus on Fuzhuan Tea,was easy to produce sexual spores when a small amount of sodium chloride was added to the MYA solid mediu,and to produce conidia when a high concentration of sodium chloride was added to the MYA solid medium.Therefore,Aspergillus cristatum could be used as a good material to study the molecular mechanism of the sexual and asexual development of filamentous fungi.In the filamentous fungus model organism Aspergillus nidulans,its asexual development was regulated by the central regulatory pathway brl A,aba A,wet A,and the central regulatory pathway modifier gene StuA,and the central regulatory pathway activator flu G and flb A-E.FlbD is an upstream developmental activator,which has a regulatory effect on asexual development.Studies have reported that while StuA gene affects asexual development,it also participates in the regulation of sexual development.In this study,Aspergillus cristatus was used as the experimental material,the FlbD and StuA genes were used as the entry point,and the Agrobacterium tumefaciens-mediated homologous recombination method and gene overexpression technology were used to study the functions of FlbD and StuA genes.The study of Flu G-Brl A,the regulation pathway of conidia formation in Aspergillus cristatus,has a positive effect,and has reference significance for the study of the molecular mechanism of filamentous fungus sporulation.The main conclusions are as follows:1.The full length of flbD gene is 951 bp,without intron sequence,encoding 316 amino acids and no transmembrane structure,conserved domain is SANT,Myb?DNA-bind?6,containing PLN03212,REB1 superfamily protein;2.The StuA gene is 1933 bp in length,encoding 610 amino acids,and the protein encoded by it has no transmembrane structure.The gene contains 2 introns,50 bp and50 bp respectively,located at 262-313 bp and 507-558 bp in StuA gene,and contains the Kil A-N conserved domain,located at 557-724 bp of StuA gene sequence;PHA03247 superfamily protein is located at 854-1636 bp of the StuA gene sequence;3.FlbD gene knockout strain was obtained by homologous recombination mediated by Agrobacterium tumefaciens.The copy number of foreign gene HYG of FlbD gene knockout strain was confirmed to be a single copy by RT qPCR.Comparing wild-type Aspergillus cristatus,the number of ?FlbD conidia is about 4.7 times lower than that of the wild type.The growth rate of ?FlbD mutant was not significantly different from that of the wild type,but it produced a large number of "cotton" shaped aerial hyphae on the medium with different concentrations of sodium chloride.Its hyphae at the edge of colony were sparse.The conidia of ?FlbD mutant was mainly concentrated in the center of the colony and were produced latter than that of the wild type on 3M sodium chloride medium for 3 days;4.After overexpression of the FlbD gene in Aspergillus cristatus,the strain will produce a large number of conidia.the number of conidia is about 4.2 times more than that of the wild type on MYA medium with 2 mol/L NaCl;the number of conidia is about 3.5 times more than that of the wild type on MYA medium with 3 mol/L NaCl;5.StuA gene knockout strain was obtained by homologous recombination mediated by Agrobacterium tumefaciens.The copy number of foreign gene HYG of StuA gene knockout strain was confirmed to be a single copy by RT qPCR.Comparing wild-type Aspergillus cristatus,the mutants did not produce capsule and conidia;On the medium with different concentrations of sodium chloride,The colony diameter of ?StuA mutant was smaller than that of wild type,and the colony was brown yellow with a large amount of dark brown exudate;6.After overexpression of StuA gene in Aspergillus cristatus,a large number of conidia were produced in the colony center,and the number of conidia was 1.7 times higher than that of wild type on Mya medium of 2 mol / L NaCl;Under the condition of 3 mol / L NaCl,its number was 1.8 times higher than that of wild type;The colony of the overexpression strain was wrinkled and produced a small amount of dark brown exudate on Mya medium with 0 mol / L NaCl.