Functional Study Of ImeB And PpgA Genes In The Formation Of Occlusive Shells In Aspergillus Cristatus

Aspergillus cristatus is the dominant fungus in the flowering process of Fuzhuan tea.The quantity of it's cleistothecia known as"golden flowers"plays an important role in the quality judgment of Fuzhuan tea.In the preliminary study,it was found that under high osmotic pressure,Aspergillus cristatus was more likely to produce pure sexual spores than other filamentous fungi.Therefore,Aspergillus cristatus is a good material to study the mechanism of sexual sporulation of filamentous fungi.As an important structure in the sexual development of Aspergillus cristatus,to study cleistothecia formation is of great significance in Fuzhuan tea production and for mechanism of sexual sporulation of filamentous fungi.In aspergillus sexual development,imeB gene plays a positive role in regulating the development of the cleistothecia.G protein-coupled receptor and MAKP pathway play a very important role in the spore production process of Aspergillus.ppgA gene as a precurs gene of pheromone responds to G protein-coupled receptor and MAKP pathway,it is speculated that these two genes might be closely related to the cleistothecia formation.In this study,strains of Ku70 preserved in the laboratory as materials,the research results are as follows:1)Based on bioinformatics analysis for the gene sequence of imeB and ppgA,the imeB gene(SI65?05226)sequence is 1542 bp in length,containing 1 intron,81 bp in length,1461 bp in m RNA sequence length,encoding 487 amino acids containing12 helical structures,and its encoded protein belongs to the MFS superfamily protein.The ppgA gene(SI65?03062)is 330bp in length without introns.The m RNA sequence is 330 bp in length and encodes 109 amino acids.2)Three ppgA knockout transformants were obtained by gene knockout technology.Morphological study showed that ppgA knockout strains possessed mycelia thicker and color light yellow colony in MYA culture medium containing different concentrations(0,1,2 mol/L NaCl)of NaCl at 28?8 days.The knockout strains produed cleistothecium only in 1 mol/L NaCl concentrations,but the cleisothecium yield is lower than the control strains.ppgA gene missing will reduce cleistothecium production of aspergillus cleistothecium.The experimental data showed that the cleistothecium amount of ppgA knockout strains was about 3/4mm~2,while that of control strains was about 44/4mm~2,and the number of cleistothecium of ppgA knockout strains was about 15 times lower than that of control strains.These results indicated that ppgA gene deletion could significantly reduce cleistothecium formation during the sexual development of Aspergillus cristatus,and ppgA gene was positively correlated with the sexual development of Aspergillus cristatus.ppgA deletion could slow down the growth rate of Aspergillus cristatus,and also had a certain effect on asexual sporulation.3)Four imeB gene knockout transformants were obtained by gene knockout technology.Morphological observation showed that imeB gene knockout strains colony was black in the center on MYA medium(37?,8 days).The colonies of imeB knockout strains and control strains were golden yellow,and a large number of cleistothecium were produced at 1mol/L NaCl concentration.At the concentration of2mol/L NaCl,aerial hyphae grew and formed an aerial mycelium layer in the culture medium center of imeB knockout strains.Conidia were produced in the center of imeB knockout strains.Both the number of cleistothecium and conidia of the imeB knockout strain decreased.4)One strain of ppgA gene overexpressing strain was obtained by gene overexpression technology.Morphological observation showed that ppgA gene overexpressing strain had a larger cleistothecium,a shorter maturity period and the ascospores increased by 2.81 times on MYA medium(5%NaCl,28?).