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Updated: Jul 15, 2025
Mating-based Overexpression Library Screening in Yeast
Published on: July 6, 2018
Screening of genes encoding proteins that interact with Nrf2: Probing a cDNA library from Mytilus coruscus using a
Li Zhu1, Yaru Li1, Longmei Qiu1
1National Engineering Research Center of Marine Facilities Aquaculture, Marine Science and Technology College, Zhejiang Ocean University, Zhoushan, Zhejiang, 316004, China.
Abstract:
The Nuclear factor Erythroid 2-related factor 2 (Nrf2) is the most important endogenous antioxidant factor in organisms, and it has been demonstrated that it exerts extensive control over the immune response by interacting with crucial innate immunity components directly or indirectly. Although Nrf2 has been widely confirmed to be involved in stress resistance in mammals and some fish, its contribution to mollusks oxidative stress resistance has not frequently been documented. In this investigation, total RNA was taken from the digestive gland of M. coruscus, and a cDNA library was constructed and screened using the GATEWAY recombination technology. The Nrf2 cDNA sequence of M. coruscus was cloned into the pGBKT7 vector to prepare the bait plasmid. Using yeast two-hybrid system, after auxotrophic medium screening, sequencing, and bioinformatics analysis, 13 binding proteins that interacted with Nrf2 were finally identified. They were QM-like protein, 40S ribosomal protein S4 (RPS4), ribosomal protein S2 (RPS2), ribosomal protein L12 (RPL12), EF1-alpha mRNA for elongation factor 1 alpha (eEF1-alpha), ferritin, alpha-amylase, trypsin, vdg3, period clock protein, cyclophilin A isoform 1 (CYP A), serine protease CFSP2, histone variant H2A.Z (H2A.Z). For a better understanding the physiological function of Nrf2 in animals and as a potential target for future research on protein roles in Nrf2 interactions, it is crucial to clarify these protein interactions.
Insights
Nuclear factor Erythroid 2-related factor 2 (Nrf2) is key for antioxidant defense. This study identifies 13 proteins interacting with Nrf2 in M. coruscus, advancing understanding of mollusk stress resistance.
Area of Science:
- Molecular Biology
- Biochemistry
- Marine Biology
Background:
- Nuclear factor Erythroid 2-related factor 2 (Nrf2) is a critical endogenous antioxidant regulator.
- Nrf2 influences immune responses and stress resistance in various species.
- Its role in mollusk oxidative stress resistance remains under-documented.
Purpose of the Study:
- To investigate the interaction partners of Nrf2 in the mollusk M. coruscus.
- To elucidate the contribution of Nrf2 to oxidative stress resistance in mollusks.
Main Methods:
- Cloning of M. coruscus Nrf2 cDNA.
- Construction of a cDNA library from digestive gland tissue.
- Yeast two-hybrid system screening and bioinformatics analysis.
Main Results:
- Identification of 13 Nrf2-interacting proteins.
- Identified proteins include ribosomal proteins, ferritin, proteases, and histone variants.
- Specific proteins identified: QM-like protein, RPS4, RPS2, RPL12, eEF1-alpha, ferritin, alpha-amylase, trypsin, vdg3, period clock protein, CYP A, serine protease CFSP2, H2A.Z.
Conclusions:
- This study reveals novel Nrf2 interactions in M. coruscus.
- These findings provide insights into Nrf2's function in mollusk stress response.
- Identified proteins serve as potential targets for future research on Nrf2's physiological roles.
