1. Trypsin Inhibitors Quantification: In this phase, 300 soybean samples from the Southern Core Collection (SCC) were analyzed for Kunitz trypsin inhibitor (KTI) and Bowman-Birk trypsin inhibitor (BBTI) concentrations. KTI concentration ranged from 0.492 to 7.675 mg/g in seeds and 0.240 to 10.756 mg/g in soybean meals, while BBTI concentrations ranged from 0.338 to 7.788 mg/g in seeds and 0.0 to 4.275 mg/g in meals. A robust NIR calibration model was developed to predict KTI and total TI concentrations using the spectral data, with an R-squared value of 0.96. This model has significant implications for improving the speed and accuracy of TI quantification in the soybean industry.
2. Protein Digestibility and Temperature Treatments: A custom-designed steam toasting system was applied to soybean meals at different temperatures (60°C, 80°C, 100°C, and 121°C). Protein digestibility improved with increasing temperature, reaching optimal levels at 100°C and 121°C. At these temperatures, the trypsin binding activity of KTI was significantly reduced. The protein solubility decreased as the temperature increased, likely due to protein aggregation at higher temperatures. SDS-PAGE analysis confirmed reduced protein complexity at 100°C and 121°C, supporting the conclusion that thermal processing effectively reduces antinutritional factors while maintaining protein integrity.
3. Gene Editing for Trypsin Inhibitor Reduction: In the most groundbreaking part of the project, researchers successfully utilized CRISPR-Cas9 genome editing to target BBTI genes responsible for trypsin inhibitor production in soybean. A comprehensive genome sequence analysis led to the identification of seven candidate BBTI genes for editing, which include Glyma09g28720, Glyma09g28700, and Glyma09g28730. These genes are highly expressed in soybean seeds and are prime targets for reducing trypsin inhibitor content through genome modification.
Researchers developed three guide RNAs to specifically target these nine BBTI genes, allowing for precise gene knockouts. The editing construct (p3399-BBTI-3gRNAs) was successfully transformed into the soybean cultivar Jack using Agrobacterium-mediated transformation. Additionally, an Agrobacterium strain carrying a Ruby reporter gene was employed to monitor transformation efficiency, with successful transformation evidenced by the appearance of reddish spots on the cotyledonary nodes. Over 23 putative transgenic soybean seedlings were identified, which will be further validated through genotyping to confirm successful BBTI knockout.
In addition, this report includes a detailed expression analysis of the lectin genes, another antinutritional factor in soybeans. Six candidate lectin genes were identified through BLAST analysis and qPCR validation, with varying levels of expression across different tissues. These genes could also be targeted for future genome editing to reduce lectin content.
4. Lectin Measurement and Its Correlation with TI: An optimized high-throughput HPLC protocol was established to measure lectin content in soybean seeds and meals, with concentrations ranging from 1.16 to 12.46 mg/g. Six soybean lines were identified that exhibit both low lectin and low KTI content, which are ideal candidates for breeding programs aimed at reducing antinutritional factors. Importantly, no correlation was observed between the activity or concentration of TIs and lectin activity, suggesting that these two factors can be addressed independently.
The comprehensive research presented in this report highlights significant progress in the mitigation of trypsin inhibitors and other antinutritional factors through innovative genome editing techniques, thermal processing, and high-throughput phenotyping. These advancements lay the groundwork for future improvements in soybean meal quality, with a strong potential for publication in high-impact journals such as Crop Science.