Screening And Verification Of MAT-interacting Proteins In Aspergillus Cristatu

The sporulation mechanism are different between Aspergillus cristatus and Aspergillus nidulans.The sporulation of Aspergillus nidulans is regulated by light,and is difficult to gain the pure sexual stage.Aspergillus cristatus owns pure sexual and asexual reproductive strategies under low-and high-osmotic pressure conditions,so it is a good material for studying sexual development.In our earlier research,the sexual production of Aspergillus cristatus was regulated by MAT1-1-1 and MAT1-2-1,but we are unclear to their regulation mechanism.The interaction of proteins is a important tool to reveal the regulation mechanism.Therefore,this study explored the interaction between MAT proteins and others to clarify the sporulation mechanism in Aspergillus cristatus,there is positive effect on improving the sporulation mechanism of Aspergilli.In this study,we studied the interaction between MAT protein and others by GST pull-down,and then the relationship was verified by Yeast two-hybrid.The results are as follows:(1)The fusion expression vectors of GST-MAT1-1-1 and GST-MAT1-2-1 were successfully constructed,and then the bait protein which can captured the interaction proteins from the total protein of Aspergillus cristatus was expressed and purified,its composition were detected by LC-MS/MS.The results showed that 56 proteins interacted with MAT1-1-1 protein,413 proteins interacted with MAT1-2-1 protein.GO analysis showed that these proteins are involved in biological processes such as translation regulation,metabolic processes,protein binding and transport,and have biological activities such as nucleotide binding,catalysis and protein binding.(2)SI65＿03348 and SI65＿00917 were screened as candidate interaction proteins,the results of the bioinformatics analysis showed that SI65＿03348 protein had no signal peptide and transmembrane structure,containing NAC superfamily domain and UBA＿NAC＿euk superfamily domain.SI65＿00917 has no signal peptide,no transmembrane structure,containing NADB conserved structural domain.(3)The expression levels of SI65＿03348 and SI65＿00917 genes was detected by RT-q PCR at different stages of the sexual development in Aspergillus cristatus.The results indicated that SI65＿03348 gene can be related to the mating recognition of mycelium,and SI65＿00917 gene can be related to the sporulation of Aspergillus cristatus.(4)The results from yeast two-hybrid showed that SI65＿00917 protein has auto-activating;meanwhile,there are the interaction between SI65＿03348 and MAT1-1-1 or MAT1-2-1 proteins.