Functional Study Of The Calcium Signalling Regulatory Gene Fig In Aspergillus Cristatus

Aspergillus cristatus was a symbolic fungus of the quality of Fuzhuan brick tea.On artificial medium,pure ascospore spores or conidia could be obtained by controlling the concentration of NaCl.In general,the fungus could respond to an osmotic pressure through the high-osmolarity glycerol(HOG)pathway.In our the laboratory,analysis of transcriptome sequencing had been done under the high-and low-osmolarity conditions of A.cristatus.It was found that HOG pathway was not activated under high osmotic pressure culture conditions,indicating that the fungus did not regulate sporulation through the HOG pathway.It was reported that the calcium signaling pathway was involved in the osmotic pressure response.It was possible that the calcium signaling pathway was involved in the osmotic response of the fungus,in which the calcium ion transporter gene Fig on the plasma membrane could transport calcium ions,indicating that this gene was related with sexual development,asexual sporulation and hyphal growth.Therefore,in this study Fig gene of A.cristatus was selected as the research object.The construction of knockout vector,Agrobacterium-mediated transformation,extracellular calcium ions and calcium ions chelating agent to mutant strain effects and other studies were carried out,the following results were obtained:(1)Fig gene was 994 bp in length,predicted it encoding 271 amino acids,contained four transmembrane regions and three introns,belonged to hydrophobin.There was a conserved motif G??GxC(n)C between the first and second transmembrane regions.Comparing with homologous Fig genes in other species of Aspergillus,it was found that the Fig gene was structurally conserved,and the conserved domain and transmembrane structure were similar;(2)The knockout vector was constructed by homologous recombination.The Fig knockout strain was obtained by Agrobacterium tumefaciens-mediated transformation.The microscopic observation of the knockout strain revealed that the morphological characteristics of the ascospores and conidia were not different from that of the wild type.But the number of spores was significantly different.Compared with the wild type,the ascospores were down-regulated by 5 times and the conidia were down-regulatedby 4 times.The reaction of knockout strain on osmotic pressure and oxidative stress was not significantly different from that of wild type;(3)It was found that under low-osmolarity conditions;,hyphal growth of the Fig knockout strain was lower and the amount of its ascospores was lower than that of the wild type,by adding extracellular calcium ions with different concentrations.The knockout strain could not recover sexual sporulation by adding calcium ions chelating agents,but its hyphal growth was consistent with the wild type.It was suggested that the Fig gene could affect hyphal growth by controlling the entry of calcium ions and participate in the regulation of sexual sporulation by affecting other pathways.Under high-osmolarity conditions,the knockout strain was more likely to develop toward sexual development than the wild type and the amount of its conidia was further decreased with the increase of calcium ion.The calcium ion chelating agent could help the knockout strain to recover asexual sporulation,indicating that under high-osmolarity conditions,the Fig gene could have a certain inhibitory effect on the absorption of calcium ions.