AtaGenix Provides Custom CHALK10 and SD1 Protein Expression and Rabbit Polyclonal Antibody Services for Rice Chalkiness Research at China National Rice Research Institute

Research Background

Grain chalkiness in rice (Oryza sativa) negatively impacts grain quality, affecting texture, milling resilience, and consumer preference. A 2025 study published in Plant Communications (DOI: 10.1016/j.xplc.2025.101354) by researchers at the State Key Laboratory of Rice Biology and Breeding identified CHALK10, an F-box protein that negatively regulates grain chalkiness by mediating ubiquitination and degradation of SEMIDWARF-1 (SD1) via the 26S proteasome pathway. CHALK10 interacts with SD1 to modulate gibberellin (GA) levels and starch metabolism, thereby affecting endosperm development and chalkiness formation.

Client Needs

The research team required: (1) high-purity recombinant CHALK10 and SD1 proteins with affinity tags for co-immunoprecipitation (co-IP) assays to validate their physical interaction in rice endosperm; (2) rabbit polyclonal antibodies against both CHALK10 and SD1 for immunoblotting in ubiquitination and degradation studies; (3) proteins and antibodies compatible with in vitro ubiquitination assay workflows; and (4) validated reagent quality sufficient for a high-impact publication in plant molecular biology.

Technical Challenges

This plant biology project presented specific challenges:

• Prokaryotic expression of plant proteins: CHALK10 and SD1 are plant-specific proteins that lack prokaryotic homologs. Achieving soluble, functional expression in E. coli required careful construct design, tag selection (MBP for CHALK10 solubility, GST for SD1 pull-down compatibility), and expression condition optimization.
• Co-IP-grade purity: Co-immunoprecipitation assays are highly sensitive to contaminants and non-specific binding. Proteins required stringent purification to minimize background while preserving tag accessibility for pull-down experiments.
• Antibody specificity in complex plant extracts: Rice protoplast extracts contain a large number of proteins, including other F-box family members and GA biosynthesis enzymes. The polyclonal antibodies had to specifically detect CHALK10 and SD1 without cross-reacting with related plant proteins.

Customized Solutions

AtaGenix designed an integrated protein + antibody workflow for this plant biology application:

• Tag-Based Protein Expression: CHALK10 was cloned into pMAL-c2X (MBP-tag) and SD1 into pGEX-6P-1 (GST-tag), expressed in E. coli Rosetta (DE3) cells optimized for rare codon usage in plant sequences. MBP-tag enhanced CHALK10 solubility; GST-tag enabled direct glutathione pull-down for co-IP workflows.
• High-Purity Affinity Chromatography: Recombinant proteins were purified by amylose resin (MBP-CHALK10) and glutathione affinity chromatography (GST-SD1), delivering high purity suitable for co-IP and in vitro ubiquitination assays with minimal non-specific background.
• Rabbit Polyclonal Antibody Production: Rabbits were immunized with purified CHALK10 and SD1 proteins. Polyclonal antibodies were affinity-purified and validated by Western Blot against rice protoplast extracts, confirming specific detection with strong signal-to-noise for both targets.
• Assay-Ready Validation & Support: AtaGenix provided technical assistance for optimizing antibody performance in immunoblotting and co-IP, including recommended dilution ratios, lysis buffer conditions, and controls for ubiquitination detection.

Research Outcomes and Impact

The high-purity recombinant proteins and specific polyclonal antibodies enabled the research team to confirm the CHALK10–SD1 physical interaction via co-IP and demonstrate that CHALK10 mediates SD1 ubiquitination and 26S proteasomal degradation. This reduces bioactive GA levels, alters starch biosynthesis gene expression during grain filling, and ultimately decreases grain chalkiness. The study established CHALK10 as a novel molecular target for improving rice grain quality through breeding, with direct implications for global food security and consumer acceptance of rice varieties.

Figure 1. Graphical overview of the CHALK10–SD1 axis. The F-box protein CHALK10 targets SD1 for ubiquitination and 26S proteasomal degradation, modulating gibberellin levels and starch metabolism to regulate rice grain chalkiness.

Figure 2. Co-immunoprecipitation and immunoblot validation of the CHALK10–SD1 interaction using AtaGenix-produced recombinant proteins and rabbit polyclonal antibodies. Specific detection confirmed direct protein-protein interaction in rice protoplast extracts.

This case study is based on a published research collaboration. Results may vary depending on target protein, expression system, and experimental conditions. All proprietary client information is subject to NDA. Reference: Plant Communications. 2025. DOI: 10.1016/j.xplc.2025.101354