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. 2025 Jun;67(6):2319-2338.
doi: 10.1007/s12033-024-01199-2. Epub 2024 Jun 2.

Construction of a Membrane Yeast Two-Hybrid Library and Screening of MsPYR1-Like Interacting Proteins in Malus sieversii

Zhen Fang  1 , Kai Zhang  1 , Jing Li  1 , Juan Ma  1 , Chunxiu Ye  2
Affiliations

Construction of a Membrane Yeast Two-Hybrid Library and Screening of MsPYR1-Like Interacting Proteins in Malus sieversii

Zhen Fang et al. Mol Biotechnol. 2025 Jun.

Abstract

To investigate the biological effects of the ABA receptor pyrabactin resistance 1-like (PYR1-like) in Malus sieversii seeds, the proteins interacting with MsPYR1-like were screened by the membrane yeast two-hybrid library based on the split-ubiquitin system, and to construct the bait vector pBT3-SUC-PYR1 for Malus sieversii cDNA library, which had no self-activating effect on the yeast cells of the pPR3-N membrane yeast two-hybrid library. The library titer assay showed that it could meet the requirements for membrane yeast two-hybrid library screening. After sequencing, GenBank database blast, and yeast rotary validation, 28 candidate proteins interacting with MsPYR1-like were obtained, including ribosomal proteins, late embryogenesis abundant proteins, F-actin-capping proteins, phytochrome-interacting proteins, low-temperature-inducible 65 kDa protein-like, senescence-associated, PP2C and SnRK2 family members, and unknown proteins. Gene ontology analysis of the interaction proteins was related to plant hormone response and negative regulation of seed germination, overexpression of MsPYR1-like in Arabidopsis negatively regulates seed germination, and the study of the biological roles of MsPYR1-like interacting proteins lays the foundation for revealing the lifting of seed dormancy in Malus sieversii.

Keywords: Malus sieversii Ledeb. Roem; MsPYR1-like; Expression analysis; Membrane yeast two-hybrid library; Protein interaction.

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Conflict of interest statement

Declarations. Conflicts of interest: The authors declare that they have no conflicts of interest.

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