Screening of Interacting Proteins of MAT in <i>Aspergillus critatus</i> by GST Pull-down

doi:10.13523/j.cb.2108070

China Biotechnology

2022, Vol. 42

Issue (3): 1-12 DOI: 10.13523/j.cb.2108070

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

Download:

HTML

PDF(6413KB) HTML
Export: BibTeX | EndNote (RIS)

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

Received: 30 August 2021 Published: 07 April 2022

ZTFLH:

Q819

Corresponding Authors: Yong-yi GE E-mail: 746560455@qq.com

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	WU Juan
	XU Ning
	ZHANG Sheng-hua
	ZHANG Xiao-dan
	LIU Yuan-yuan
	GE Yong-yi

Cite this article:

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.

URL:

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

Fig.1 Construction of fusion expression carrier (a) pET-GST-MAT1-1-1 plasmid digested by Xba I, M: DNA marker; 1: recombinant plasmid digestion; 2: plasmid DNA (b) pET-GST-MAT1-2-1 plasmid digested by Nco I, M: DNA marker; 1: recombinant plasmid digestion; 2: plasmid DNA

Fig.2 Expression and purification of the fusion protein (a) SDS-PAGE electrophoresis of broken supernatant and pellet of pET-GST-MAT1-1-1, 1:the supernatant protein; 2:insoluble protein(the red arrow is a recombinant protein) (b) SDS-PAGE electrophoresis of purified fusion protein, 1:target protein(the red arrow is a recombinant protein) (c) SDS-PAGE electrophoresis of pET-GST-MAT1-2-1,1:GST-MAT1-2-1 supernatant recombinant protein purification

Fig.3 GST-MAT1-1-1 pull-down result detection (a)Western blot detection before GST-MAT1-1-1 pull-down (b) Western blot detection after GST-MAT1-1-1 pull-down (c) Silver stain detection after GST-MAT1-1-1 pull-down

Fig.4 GST-MAT1-2-1 pull-down result detection (a)Western blot detection before GST-MAT1-2-1 pull-down (b)Western blot detection after GST-MAT1-2-1 pull-down (c) Silver stain detection after GST-MAT1-2-1 pull-down

Fig.5 Proteins identified by CTRL, EX protein samples in the MAT1-1-1(a) and MAT1-2-1(b)

Table 1 The information table of interaction protein with MAT1-1-1

Table 2 The information table of interaction protein with MAT1-2-1

Fig.6 MAT1-1-1 (a), MAT1-2-1 (b)reciprocal protein GO function analysis (cell composition, biological process, molecular function)

Fig.7 RNA extraction and retrorecorded PCR electrophoresis (a) Morphological characteristics of Aspergillus cristatus in three different sexual development stages (b) RNA from the different sexual development of Aspergillus critatus,M: DNA maker; 1: RNA during the nutritional growth stage(S1); 2: RNA during the formation of closed shell(S2); 3:RNA during ascospore formation period(S3) (c) The detection result of GAPDH, M: DNA marker; 1~6: S1 stage; 7~12:S2 stage; 13~18:S3 stage

Table 3 Cq and F values of the SI65_03348, SI65_00917 and GAPDH genes at the different stages

Fig.8 Relative expression index of the SI65_00917, SI65_03348 and GAPDH genes at different periods (a) The multiples of expression difference between SI65_00917 and GAPDH at different stages of sexual development (b) The multiples of expression difference between SI65_04438 and GAPDH at different stages of sexual development

Fig.9 Yeast two-hybrid vector construction (a) CDS sequence amplification, laneM: DNA maker, 1: cds sequence amplification of MAT1-1-1(1.1 kb); 2:cds sequence amplification of MAT1-2-1(1.0 kb); 3:cds sequence amplification of SI65_00917(0.8kb); 4: cds sequence amplification of SI65_03348(0.6 kb) (b) Recombinant vector double restriction digestion verification, M: DNA maker; 1: AD-MAT1-1-1 plasmid digested by EcoR I and Nde I; 2: AD-MAT1-2-1 plasmid digested by EcoR I and Nde I; 3: BD-00917 plasmid digested by EcoR I and BamH I; 4: BD-03348 plasmid digested by EcoR I and BamH I

Fig.10 Identification of auto-activation and interaction verification (a) Identification of auto-activation for bait BD-00917, BD-03348 (b) Interaction verification of SI65_03348 and MAT1-1-1, MAT1-2-1


[1]	Ene I V, Bennett R J. The cryptic sexual strategies of human fungal pathogens. Nature Reviews Microbiology, 2014, 12(4):239-251. doi: 10.1038/nrmicro3236

[2]	Whittle C A, Nygren K, Johannesson H. Consequences of reproductive mode on genome evolution in fungi. Fungal Genetics and Biology, 2011, 48(7):661-667. doi: 10.1016/j.fgb.2011.02.005 pmid: 21362492

[3]	Szewczyk E, Krappmann S. Conserved regulators of mating are essential for Aspergillus fumigatus cleistothecium formation. Eukaryotic Cell, 2010, 9(5):774-783. doi: 10.1128/EC.00375-09 pmid: 20348388

[4]	Ni M, Feretzaki M, Sun S, et al. Sex in fungi. Annual Review of Genetics, 2011, 45(1):405-430. doi: 10.1146/genet.2011.45.issue-1

[5]	Liu K H, Shen W C. Mating differentiation in Cryptococcus neoformans is negatively regulated by the Crk1 protein kinase. Fungal Genetics and Biology, 2011, 48(3):225-240. doi: 10.1016/j.fgb.2010.11.005

[6]	Heitman J, Sun S, James T Y. Evolution of fungal sexual reproduction. Mycologia, 2013, 105(1):1-27. doi: 10.3852/12-253 pmid: 23099518

[7]	施笑笑, 王教瑜, 王艳丽, 等. 子囊菌交配型位点与交配型基因研究进展. 微生物学通报, 2020, 47(5):1572-1581.

[7]	Shi X X, Wang J Y, Wang Y L, et al. Mating type genes in ascomycetes: a review. Microbiology China, 2020, 47(5):1572-1581.

[8]	Turgeon B G, Yoder O C. Proposed nomenclature for mating type genes of filamentous ascomycetes. Fungal Genetics and Biology, 2000, 31(1):1-5. pmid: 11118130

[9]	Ge Y Y, Yu F M, Yang Z J, et al. Genetic basis and function of mating-type genes in Aspergillus cristatus. Mycosphere, 2019, 10(1):622-633. doi: 10.5943/mycosphere

[10]	Grognet P, Bidard F, Kuchly C, et al. Maintaining two mating types: structure of the mating type locus and its role in heterokaryosis in Podospora anserina. Genetics, 2014, 197(1):421-432. doi: 10.1534/genetics.113.159988 pmid: 24558260

[11]	Kanamori M, Kato H, Yasuda N, et al. Novel mating type-dependent transcripts at the mating type locus in Magnaporthe oryzae. Gene, 2007, 403(1-2):6-17. pmid: 17881155

[12]	Yong M L, Yu J J, Pan X Y, et al. Two mating-type genes MAT1-1-1 and MAT1-1-2 with significant functions in conidiation, stress response, sexual development, and pathogenicity of rice false smut fungus Villosiclava virens. Current Genetics, 2020, 66(5):989-1002. doi: 10.1007/s00294-020-01085-9

[13]	Becker K, Beer C, Freitag M, et al. Genome-wide identification of target genes of a mating-type α-domain transcription factor reveals functions beyond sexual development. Molecular Microbiology, 2015, 96(5):1002-1022. doi: 10.1111/mmi.2015.96.issue-5

[14]	Dyer P S, Paoletti M. Reproduction in Aspergillus fumigatus: sexuality in a supposedly asexual species. Medical Mycology, 2005, 43(S1):S7-S14.

[15]	Böhm J, Hoff B, O’Gorman C M, et al. Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum. PNAS, 2013, 110(4):1476-1481. doi: 10.1073/pnas.1217943110

[16]	Arnaise S, Debuchy R, Picard M. What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina. Molecular and General Genetics: MGG, 1997, 256(2):169-178.

[17]	Kim H, Wright S J, Park G, et al. Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa. Genetics, 2012, 190(4):1389-1404. doi: 10.1534/genetics.111.136358

[18]	Whiteway M S, Wu C, Leeuw T, et al. Association of the yeast pheromone response G protein beta gamma subunits with the MAP kinase scaffold Ste5p. Science, 1995, 269(5230):1572-1575. pmid: 7667635

[19]	Chol K Y, Satterberg B, Lyons D M, et al. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell, 1994, 78(3):499-512. doi: 10.1016/0092-8674(94)90427-8

[20]	任春光, 谭玉梅, 任秀秀, 等. 冠突曲霉veA基因缺失型与野生型的差异代谢物研究. 菌物学报, 2018, 37(2):193-204.

[20]	Ren C G, Tan Y M, Ren X X, et al. Differential metabolite analysis of the veA gene deletion and wild type strains of Aspergillus cristatus. Mycosystema, 2018, 37(2):193-204.

[21]	余春芳, 熊庆, 李文仿, 等. 冠突散囊菌nsdD基因超表达菌株的构建及表型分析. 基因组学与应用生物学, 2017, 36(3):900-905.

[21]	Yu C F, Xiong Q, Li W F, et al. Construction and phenotypic analysis of over-expression strain of nsdD gene in Eurotium cristatum. Genomics and Applied Biology, 2017, 36(3):900-905.

[22]	Lehti-Shiu M D, Panchy N, Wang P P, et al. Diversity, expansion, and evolutionary novelty of plant DNA-binding transcription factor families. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2017, 1860(1):3-20. doi: 10.1016/j.bbagrm.2016.08.005

[23]	Nolting N, Pöggeler S. A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora. Eukaryotic Cell, 2006, 5(7):1043-1056. pmid: 16835449

[24]	Jacobsen S, Wittig M, Pöggeler S. Interaction between mating-type proteins from the homothallic fungus Sordaria macrospora. Current Genetics, 2002, 41(3):150-158. pmid: 12111096

[25]	郑欣欣. 茯砖茶中“金花”菌产孢机制及其功能性研究. 西安: 陕西科技大学, 2015.

[25]	Zheng X X. Study on sporulation mechanism and function of ‘Jinhua’ fungi in fuzhuan brick tea. Xi’an: Shanxi University of Science & Technology, 2015.

[26]	Rao X Y, Huang X L, Zhou Z C, et al. An improvement of the 2-ΔΔCt method for quantitative real-time polymerase chain reaction data analysis . Biostatistics,Bioinformatics and Biomathematics, 2013, 3(3):71-85.

[27]	Wissmueller S, Font J, Liew C W, et al. Protein-protein interactions: analysis of a false positive GST pulldown result. Proteins, 2011, 79(8):2365-2371. doi: 10.1002/prot.23068

[1]	JIA Gui-yan,WANG Yong-jie,CHEN Zhi-kang,CHEN Xing,YIN Kui-de,LI Wen,WANG Yan-hong. *Cloning and Analysis of Novel Functional Genes in Halomonas alkaliphila* DSM 16354 ^T**[J]. China Biotechnology, 2022, 42(3): 27-37.

[2]	XU Hui, DING Jian, SHI Zhong-ping. *Research on a Fed-batch of Nitrogen Source Fermentation Process to Improve the Spores of Bacillus Licheniformis* BF-002**[J]. China Biotechnology, 2022, 42(3): 47-54.

[3]	WU Dong,XIAO Feng,YUAN Min,CHEN Yu-bao,ZHANG Hong-xiang. Transformation of Scientific and Technological Achievements in Biomedical Field in China from 2011 to 2020[J]. China Biotechnology, 2022, 42(1/2): 191-201.

[4]	MA Ning,WANG Han-jie. Advances of Optogenetics in the Regulation of Bacterial Production[J]. China Biotechnology, 2021, 41(9): 101-109.

[5]	ZHANG Heng,LIU Hui-yan,PAN Lin,WANG Hong-yan,LI Xiao-fang,WANG Tong,FANG Hai-tian. Research Strategy for Biosynthesis of Gamma Aminobutyric Acid[J]. China Biotechnology, 2021, 41(8): 110-119.

[6]	LI Kai-xiu,SI Wei. Progress in the Treatment of Inflammatory Bowel Diseases by Exosomes Derived from Mesenchymal Stem Cells[J]. China Biotechnology, 2021, 41(7): 66-73.

[7]	WANG Yu-xuan,CHEN Ting,ZHANG Yong-liang. Research Progress on the Biological Function of MiR-148[J]. China Biotechnology, 2021, 41(7): 74-80.

[8]	BAI Jia-cheng,CHI Ming-zhe,HU Ya-wen,HAO Meng,ZHANG Xue-lian. Construction and Biological Characteristics of ClpC and ClpX Knock-down Strains in Mycobacterium smegmatis[J]. China Biotechnology, 2021, 41(6): 13-22.

[9]	ZHANG Ling,CAO Xiao-dan,YANG Hai-xu,LI Wen-lei. The Application of Continuous Purification in Affinity Chromatography and Evaluation of Production Scale-up[J]. China Biotechnology, 2021, 41(6): 38-44.

[10]	ZHANG Sai,WANG Gang,LIU Zhong-ming,LI Hui-jun,WANG Da-ming,QIAN Chun-gen. Development and Performance Evaluation of a Rapid Antigen Test for SARS-CoV-2[J]. China Biotechnology, 2021, 41(5): 27-34.

[11]	DUAN Yang-yang,ZHANG Feng-ting,CHENG Jiang,SHI Jin,YANG Juan,LI Hai-ning. The Effect of SIRT2 on Apoptosis and Mitochondrial Function in Parkinson’s Disease Model Cells Induced by MPP⁺[J]. China Biotechnology, 2021, 41(4): 1-8.

[12]	WANG Yi-han,LI Hai-yan,XUE Yong-chang. The Structural Characteristics and Engineering Reconstruction of Flavin-dependent Halogenase[J]. China Biotechnology, 2021, 41(4): 74-80.

[13]	HU Sheng-tao,ZHANG Er-bing,LIN Ye,ZHANG Feng,HUANG Dan,SONG Hou-pan,LIU Bin,CAI Xiong. Research Advances on the Therapy of Rheumatoid Arthritis with the Nanotechnology Based on Transdermal Drug Delivery System[J]. China Biotechnology, 2021, 41(2/3): 98-106.

[14]	HE Wei,ZHU Lei,LIU Xin-ze,AN Xue-li,WAN Xiang-yuan. Research Progress on Maize Genetic Transformation and Commercial Development of Transgenic Maize[J]. China Biotechnology, 2021, 41(12): 13-23.

[15]	WU Han-rong,WANG Ying,HUANG Ying-ming,LI Dong-xue,LI Zhi-fei,FANG Zi-han,FAN Lin. Promote the Innovation and Transformation of Biotechnology by Base Platform[J]. China Biotechnology, 2021, 41(12): 141-147.

Viewed

Full text

Abstract

Cited

Shared

Discussed