冠突曲霉UBC与MAT1-2-1互作关系验证及功能研究

Verification of interaction between UBC and MAT1-2-1 in Aspergillus cristatus and the functional study

【目的】验证冠突曲霉泛素结合酶(UBC)与交配型蛋白(MAT1-2-1)的互作关系,对UBC基因进行功能研究,为深入解析冠突曲霉有性产孢机制提供理论参考。【方法】采用实时荧光定量PCR检测UBC基因在冠突曲霉有性发育阶段的表达水平,酵母双杂交试验验证MAT1-2-1与UBC的互作关系,利用无缝克隆方法构建pDHt/sk-hyg-UBC过表达载体,并转化冠突曲霉获得UBC过表达菌株,对过表达菌株进行表达量检测、形态学观察及氧和盐胁迫的耐受性测定。【结果】PCR克隆获得UBC基因的编码区(CDS)序列,长度为474 bp,编码157个氨基酸残基,相对分子质量为39587.24 Da,理论等电点为5.16,为疏水的不稳定蛋白,含跨膜结构域,定位于细胞核,属于UBCc超家族成员。有性发育阶段(闭囊壳形成期和子囊孢子大量形成期)UBC基因的相对表达量显著高于营养菌丝体阶段(P<0.05,下同)。自激活检测结果显示,UBC无自激活作用;酵母双杂交验证结果显示,UBC与MAT1-2-1存在互作关系。通过构建过表达载体pDHt/sk-hyg-UBC成功获得UBC过表达菌株(OE::UBC)。与野生型菌株相比,UBC和MAT1-2-1基因在UBC过表达菌株的相对表达量均显著升高。UBC过表达菌株与野生型菌株在MYA、MYA+5%NaCl和MYA+17%NaCl固体培养基上的菌落形态无明显差异。过表达菌株能分别在含18 mmol/L过氧化氢和25%NaCl的培养基中生长,而野生型菌株不能生长,说明UBC基因的过表达提高冠突曲霉对氧胁迫和盐胁迫的耐受性。【结论】UBC基因可能与冠突曲霉的有性发育相关,且与MAT1-2-1基因存在共表达特征。由于UBC与MAT1-2-1存在互作关系,且UBC基因的过表达可明显增强冠突曲霉的抗氧化和抗盐能力,故推测UBC通过与MAT1-2-1发生互作共同参与冠突曲霉的氧胁迫和盐胁迫应答过程。

【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 develop‐ment stage of A.cristatus.The interaction between MAT1-2-1 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 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 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.