Construction of a Yeast Two-Hybrid cDNA Library from Soybean Seeds at Different Developmental Stages and Screening of SW16.1 Interacting Proteins

Seed weight represents a critical yield-associated quantitative trait in soybean (Glycine max) and is governed by polygenic regulatory networks. Although several seed-weight-related genes have been functionally characterized, the interactome of the major seed-weight determining gene SW16.1 remains largely unexplored. In this study, we constructed a yeast two-hybrid (Y2H) cDNA library using pooled total RNA isolated from developing seeds of the soybean cultivar Nannong 1138-2 covering five key seed-development stages. Using GmSW16.1 (cultivated Nannong 1138-2 allelic) as bait, Y2H screening was performed. Following multi-round screening, Sanger sequencing, and filtering based on functional annotation, 11 candidate interactors assigned to nine distinct functional categories were recovered. Interactions were further verified through point-to-point Y2H assays, quantitative α-galactosidase (α-Gal) activity measurements, and bimolecular fluorescence complementation (BiFC). We confirmed that metallothionein-like protein 4A-related Glyma.18G157800 (GmMT4) and the papain-like cysteine protease Glyma.08G116300 physically interact with both GmSW16.1 and GsSW16.1 (wild soybean N24852). A two-glutamine polymorphism within the PolyQ (polyglutamine) repeat tract of these two allelic proteins did not abolish protein–protein interactions, yet significantly altered their interaction strength toward the above-mentioned partners. Glyma.08G116300 displayed preferential transcript accumulation during soybean seed development, and its transcript abundance differed dynamically between the chromosome segment substitution line CSSL3068 and its cultivated parent Nannong 1138-2 across developmental stages. Heterologous overexpression of this gene in Arabidopsis thaliana significantly increased the 1000-seed weight (by 12–15%) and increased seed length and width. This study delivers a seed-development-specific Y2H library resource for soybean research. We put forward a testable working hypothesis describing a dual-layer regulatory module integrating transcriptional control and direct protein–protein interaction for the GmSW16.1-GmMT4 regulatory node. These findings advance our mechanistic understanding of how natural PolyQ allelic variation shapes soybean seed-weight performance and provides candidate gene materials for soybean molecular design breeding.