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】 Realtime fluorescence quantitative PCR was used to detect the expression level of the UBC gene during the sexual development stage of A. cristatus. The interaction between MAT1-2-1 and UBC was verified through a yeast two-hybrid experiment. 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 157amino acid residues with a molecular weight of 39587.24 Da and an isoelectric point of 5.16. It was a hydrophobic, unstable 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 expression levels of UBC gene were significantly higher than those during the vegetative mycelium stage（P＜0.05, the same below）. Autoactivation tests showed that UBC did not self-activate, while yeast two-hybrid experiment confirmed an interaction 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 differences 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 hydrogen 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 overexpression greatly enhances the antioxidant and salt tolerance of A. cristatus. Therefore, UBC may participate in the oxidative stress and salt stress response processes of A. cristatus by interacting with MAT1-2-1.