Verification of interaction between UBC and MAT1-2-1 in Aspergillus cristatus and the functional study
[Objective]The purpose of the study was to verify the interaction between ubiquitin-conjugating enzymes(UBC)and the mating-type protein(MAT1-2-1)in Aspergillus cristatus,and to investigate the function of UBC gene,which provided a theoretical reference for elucidating the sexual sporulation mechanism of A.cristatus.[Method]Real-time fluorescence quantitative PCR was used to detect the expression level of the UBC gene during the sexual develop-ment stage of A.cristatus.The interaction between MAT 1-2-1 and UBC was verified through a yeast two-hybrid experi-ment.The pDHt/sk-hyg-UBC overexpression vector was constructed via seamless cloning and subsequently transformed into A.cristatus to generate a UBC overexpression strain.The expression levels of UBC,morphological characteristics,and oxidative and salt stress tolerance were observed and analyzed in both the overexpression strains.[Result]The results showed that PCR cloning obtained the coding region(CDS)sequence of UBC gene,measuring 474 bp and encoding 157 amino acid residues with a molecular weight of 39587.24 Da and an isoelectric point of 5.16.It was a hydrophobic,un-stable protein with a transmembrane domain and was located in the nucleus,belonging to the UBCc superfamily.During the sexual development stages(cleistothecium formation and massive ascospore formation stages),the relative expres-sion levels of UBC gene were significantly higher than those during the vegetative mycelium stage(P＜0.05,the same be-low).Autoactivation tests showed that UBC did not self-activate,while yeast two-hybrid experiment confirmed an inter-action between UBC and MAT1-2-1.The pDHt/sk-hyg-UBC overexpression vector was successfully constructed,and the UBC overexpression strain(OE∷UBC)was obtained.Compared to the wild-type strain,the relative expression levels of both UBC and MAT1-2-1 genes were significantly increased in the UBC overexpression strain.There were no great differen-ces in colony morphology between the UBC overexpression and wild-type strains in MYA,MYA+5％NaCl,and MYA+17％NaCl solid medium.The overexpression strain was capable of growing in the medium with 18 mmol/L of hy-drogen peroxide and 25％of NaCl,while the wild-type strain could not,indicating that overexpression of the UBC gene enhanced the oxidative and salt stress tolerance of A.cristatus.[Conclusion]The UBC gene is likely related to the sexual development of A.cristatus and exhibits co-expression with MAT1-2-1 gene.UBC interacts with MAT1-2-1,and its over-expression greatly enhances the antioxidant and salt tolerance of A.cristatus.Therefore,UBC may participate in the oxida-tive stress and salt stress response processes of A.cristatus by interacting with MAT1-2-1.
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