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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (3): 1-12    DOI: 10.13523/j.cb.2108070
研究报告     
GST pull-down筛选冠突曲霉MAT的互作蛋白*
吴娟,徐宁,张胜花,张小丹,刘缘圆,葛永怡**()
贵州大学生命科学学院 贵阳 550025
Screening of Interacting Proteins of MAT in Aspergillus critatus by GST Pull-down
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
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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。
关键词： 冠突曲霉;  MAT;  GST pull-down;  互作蛋白    
Abstract:

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 A.cristatus through direct or indirect interaction with the other proteins.
Key words: Aspergillus critatus    MAT    GST pull-down    Interaction protein
收稿日期: 2021-08-30 出版日期: 2022-04-07
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(31860022);贵州省科技计划资助项目(黔科合基础20201Y056)
通讯作者: 葛永怡     E-mail: 746560455@qq.com
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吴娟
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张胜花
张小丹
刘缘圆
葛永怡

引用本文:

吴娟, 徐宁, 张胜花, 张小丹, 刘缘圆, 葛永怡. GST pull-down筛选冠突曲霉MAT的互作蛋白*[J]. 中国生物工程杂志, 2022, 42(3): 1-12.

WU Juan, XU Ning, ZHANG Sheng-hua, ZHANG Xiao-dan, LIU Yuan-yuan, GE Yong-yi. Screening of Interacting Proteins of MAT in Aspergillus critatus by GST Pull-down. China Biotechnology, 2022, 42(3): 1-12.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2108070    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I3/1

图1  融合表达载体构建
图2  融合蛋白的表达纯化
图3  GST-MAT1-1-1 pull-down结果检测
图4  GST-MAT1-2-1 pull-down结果检测
图5  MAT1-1-1(a)和MAT1-2-1(b)实验中对照组、实验组蛋白样品鉴定到的蛋白质
Protein ID Gene name Coverage /% Mass /Da Unique peptide Identified by
A0A1B0THQ4_9EURO MAT1-1-1 60.420 000 553 131 1 42 804.1 2 GST
A0A1B0THQ4_9EURO MAT1-1-1 60.420 000 553 131 1 42 804.1 48 GST-MAT1-1-1
A0A1E3BR33_9EURO SI65_01009 20.649 999 380 111 7 49 950.2 8
A0A1E3BQW9_9EURO SI65_00917 10.029 999 911 785 1 31 192.2 2
A0A1E3BH40_9EURO SI65_03348 19.609 999 656 677 2 21 912 3
表1  MAT1-1-1部分互作蛋白相关信息表
Protein ID Gene name Coverage /% Mass /Da Unique peptide Identified by
A0A1E3BR33_9EURO SI65_01009 7.174 000 144 004 82 49 950.2 4 GST
A0A1B0THQ3_9EURO MAT1-2-1 17.090 000 2121 92 5 39 254.7 4
A0A1B0THQ3_9EURO MAT1-2-1 73.669 999 837 875 4 39 254.7 80 GST-MAT1-2-1
A0A1E3BKS5_9EURO SI65_02352 37.250 000 238 418 6 59 636.7 20
A0A1E3BQW9_9EURO SI65_00917 3.805 999 830 365 18 31 192.2 2
A0A1E3BH40_9EURO SI65_03348 32.350 000 739 097 6 21 912 4
表2  MAT1-2-1部分互作蛋白相关信息表
图6  MAT1-1-1(a)、MAT1-2-1(b)互作蛋白GO功能分析(细胞组成、生物过程、分子功能)
图7  冠突曲霉有性发育不同时期RNA提取及反转录后PCR电泳检测
时期 基因 Cq值 F
营养菌丝 GAPDH 14.91 1.00
SI65_03348 16.63 1.00
SI65_00917 24.28 1.00
闭囊壳形成 GAPDH 17.59 -
SI65_03348 20.31 0.50
SI65_00917 22.42 23.37
子囊孢子大量形成 GAPDH 18.41 -
SI65_03348 21.14 0.49
SI65_00917 20.33 174.85
表3  SI65_03348、SI65_00917和GAPDH基因不同时期的Cq值和F值
图8  SI65_00917、SI65_03348和GAPDH基因不同时期相对表达指数
图9  酵母双杂交载体构建
图10  诱饵载体自激活检测及互作验证
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