摘要
目的:冠突曲霉(Aspergillus cristatus)是一种同宗结合菌,它的产孢受渗透压调控,与构巢曲霉的光调控产孢机制存在较大差异。冠突曲霉的有性生殖主要受MAT1-1-1和MAT1-2-1调控,但MAT基因对该菌有性生殖的调控机制仍不清楚。期望筛选得到冠突曲霉MAT的互作蛋白,为深入研究冠突曲霉有性产孢机制奠定基础。方法:利用GST pull-down联合液相色谱-串联质谱(LC-MS/MS)技术筛选可能与冠突曲霉MAT1-1-1和MAT1-2-1互作的蛋白,结合ProteinPilot和冠突曲霉基因组注释结果进行互作蛋白的注释及GO分析,其中互作蛋白SI65_00917和SI65_03348利用RT-qPCR探索它们与有性发育的联系,并利用酵母双杂交技术初步验证它们与MAT蛋白的互作关系。结果:成功构建了GST-MAT1-1-1、GST-MAT1-2-1表达载体,诱导表达纯化出目的诱饵蛋白,分别利用诱饵蛋白捕获冠突曲霉总蛋白中的互作蛋白,经分析、筛选共鉴定出与MAT1-1-1互作的蛋白56个,与MAT1-2-1互作的蛋白413个。GO分析表明,这些蛋白参与翻译调控、代谢过程、蛋白质转运及蛋白结合等生物学过程,具有核苷酸结合活性、催化活性、蛋白结合活性;RT-qPCR结果表明互作蛋白SI65_00917可能与有性发育相关。酵母双杂交结果表明,SI65_00917蛋白具有自激活作用,可能是转录因子;SI65_03348蛋白与MAT1-1-1、MAT1-2-1在酵母中均有互作。结论:MAT通过与其他蛋白直接或间接的相互作用调控其有性发育过程。
Objective:Aspergillus cristatus is a homothallic fungus,whose sporulation is regulated by osmotic pressure,which is quite different from the light-regulated sporulation mechanism of Aspergillus nidulans.The sexual reproduction of A.cristatus is mainly regulated by MAT1-1-1 and MAT1-2-1,but the regulation mechanism of the MAT gene on the sexual reproduction is still unclear.This study aims to screen the interaction proteins of A.cristatum MAT,and lay the foundation for the further study of the sexual sporulation mechanism of A.cristatum.Methods:This study screened the interaction protein with MAT1-1-1 and MAT1-2-1 by GST pull-down combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS).We analyzed the biological information of the interaction protein using ProteinPilot,Gene Ontology and the genome databank of A.cristatus.The study also detected the expression level of SI65_00917 and SI65_03348 in sexual development by RT-qPCR,and used yeast two-hybrid technology to verify their interaction with MAT protein.Results:The GST-MAT1-1-1 and GST-MAT1-2-1 vectors were successfully constructed,and the target bait proteins were induced to express and purify.The bait proteins were used to capture the interacting proteins from the total protein of A.cristatus.The results showed that 56 proteins interacted with MAT1-1-1,and 413 proteins interacted with MAT1-2-1,respectively.GO analysis shows that these interaction proteins are involved in translation regulation,metabolic processes,protein transport,protein binding and other biological processes,and share nucleotide binding activity,catalytic activity,and protein binding activity.The results from RT-qPCR indicated that the interaction proteins SI65_00917 would participate in the sexual development in A.cristatus.Yeast two-hybrid results show that SI65_00917 protein has auto-activation and may be a transcription factor,and SI65_03348 protein interacts with MAT1-1-1 and MAT1-2-1 in yeast.Conclusion:These results indicate that MAT regulates the sexual development of
作者
吴娟
徐宁
张胜花
张小丹
刘缘圆
葛永怡
WU Juan;XU Ning;ZHANG Sheng-hua;ZHANG Xiao-dan;LIU Yuan-yuan;GE Yong-yi(School of Life Sciences,Guizhou University,Guiyang 550025,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2022年第3期1-12,共12页
China Biotechnology
基金
国家自然科学基金(31860022)
贵州省科技计划(黔科合基础20201Y056)资助项目。
