Xiaofang Zhong

Genome editing is becoming an important tool for crop improvement by enabling precise modification of genetic variation. CRISPR-Cas technologies have expanded beyond nuclease-induced mutations to include base and prime editing, transcriptional and epigenetic regulation, multiplex editing, organelle editing and targeted DNA insertion. These advances support the development of agronomically valuable alleles and extend crop improvement beyond conventional selection, recombination and random mutagenesis. Applications now include plant architecture, yield-related traits, resistance to biotic and abiotic stresses, seed composition and nutritional quality, and extend to de novo domestication, haploid induction and hybrid breeding. Current research is increasingly moving from single-gene modification toward the engineering of promoters, cis-regulatory elements, quantitative alleles and multiple-gene editing, particularly in polyploid crops. Wider breeding applications will depend on efficient delivery and regeneration, recovery of transgene-free and heritable edited germplasm, reliable targeted insertion and validation across relevant environments. Integration with pangenomics, multi-omics, phenomics, artificial intelligence and predictive breeding may further improve target identification, allele design and prediction of editing outcomes, thereby supporting the development of more precise and efficient crop-breeding strategies.

Soybean is rich in protein and oil and serves as the most important legume crop globally. Soybean mosaic virus (SMV) is a severe threat to soybean production worldwide. MutMap, a gene-mapping technology based on map-based cloning and whole-genome resequencing, is utilized to clone key regulatory genes for agronomic traits in plants. However, no relevant studies have reported the cloning of genes resistant to SMV. We used an M3 mutant population derived from ethyl methanesulfonate mutagenesis of Williams 82, and conducted field inoculation experiments involving the SMV-SC3 strain. After field validation, two lines with high resistance to SMV were finally identified. Using MutMap, we initially screened candidate genes for SMV resistance and found that the G-to-A transitions of one candidate resistance gene, Glyma.13G194900, were at base positions 122 and 166. These transitions resulted in the substitution of glycine with glutamic acid (GGA→GAA) and valine with aspartic acid (GTT→GAT), respectively. Transgenic functional validation in soybean showed that the mutant allele of Glyma.13G194900 (designated Glyma.13G194900^M) substantially enhanced resistance to SMV-SC3, in contrast to the wild-type allele, which did not enhance resistance. Our results demonstrate that MutMap can rapidly identify SMV resistance-related genes to provide a genetic resource that accelerates the breeding of new SMV-resistant soybean.

Soybean seeds show great potential as a safe and cost-effective host for the large-scale production of biopharmaceuticals and industrially important macromolecules. However, the yields of desired recombinant proteins in soybean seeds are usually lower than the economic threshold for their potential commercialization. Our previous study demonstrated that polypeptide fusion such as maize γ-zein or elastin-like polypeptide (ELP) could significantly increase the accumulation of foreign proteins. In the present study, a recombination strategy of polypeptide fusions (γ-zein or ELP) and suppression of intrinsic storage proteins (glycinin or conglycinin) via RNA interference was further exploited to improve the yield of the target protein in soybean seeds. Transgenic soybean plants harboring both polypeptide-fused green fluorescent protein (GFP) and glycinin/conglycinin RNAi expression cassettes were generated and confirmed by molecular analysis. The results showed that on both the glycinin and conglycinin suppression backgrounds, the average accumulation levels of recombinant zein-GFP and GFP-ELP proteins were significantly increased as compared to that of their counterparts without such suppressions in our previous study. Moreover, zein-GFP and GFP-ELP accumulation was also remarkably higher than unfused GFP on the glycinin suppression background. However, no significant differences were detected in the glycinin or conglycinin suppression backgrounds for the same polypeptide fusion constructs, though suppression of one of the storage proteins in soybean seeds led to a significant increase in the other. Additionally, the increases in the recombinant protein yield did not affect the total protein content and the protein/oil ratio in soybean seeds. Taken together, the results indicate that both the fusion of the foreign protein with polypeptide tags together with the depletion of endogenous storage proteins contributed to a higher accumulation of the recombinant proteins without affecting the total protein content or the protein/oil ratio in soybean seeds.

Background: Soybean seeds show great potential as a safe and cost-effective host for the large-scale production of biopharmaceuticals and industrially important macromolecules. However, the yields of desired recombinant proteins in soybean seeds are usually lower than the economic threshold (1% of total soluble protein, TSP) for potential commercialization. Here, a recombination strategy of the polypeptide (maize γ-zein, or elastin-like polypeptide, ELP) fusions and suppression of endogenous storage proteins (glycinin or conglycinin) via RNA interference (RNAi) was exploited to improve yield of the target protein in soybean seeds. Results: Transgenic soybean plants harboring both the polypeptide-fused GFP and glycinin/conglycinin RNAi expression cassettes were generated and confirmed by molecular analysis. On the glycinin suppression background, average accumulation levels of recombinant zein-GFP and GFP-ELP proteins were significantly increased as compared to that of the unfused GFP, reaching to 2.69% and 2.30% TSP in the immature seeds, and 3.49% and 4.31 % TSP in the mature seeds, respectively. However, no significant differences were detected in the glycinin or conglycinin suppression backgrounds for the same polypeptide fusion constructs, though suppression of one of the storage proteins in soybean seeds led to a significant increase of the other. Moreover, the increases in the recombinant protein yield did not affect the total protein content or the protein/oil ratio in soybean seeds Conclusions: These results demonstrate that the fusion of the foreign protein with polypeptide tags together with the depletion of endogenous storage proteins can significantly increase the expression of recombinant proteins without affecting the total protein content or the protein/oil ratio in soybean seeds.

Genomic insertions and flanking regions of transgenes in host genomes constitute a critical component of precise molecular characterization and event-specific detection, which are required in the development and assessment for regulatory approval of genetically modified (GM) crops. Previously, we reported three transgenic soybean events harboring the inverted repeats of the soybean mosaic virus NIb (nuclear inclusion b) gene, exhibiting significantly enhanced resistance to multiple Potyvirus strains. To facilitate safety assessment and event-specific detection, we identified the transgene insertion sites and flanking sequences of the events L120, L122, and L123 using whole-genome sequencing. More than 14.48 Gb sequence data (13 × coverage) were generated using the Illumina HiSeq Xten platform for each event. The sequence reads corresponding to boundaries of inserted T-DNA, and associated native flanking sequences were identified by bioinformatic comparison with the soybean reference genome (Wm82.a2.v1) and the transformation vector sequence. The results indicated that two T-DNA insertions occurred in L120, on Chr07 and Chr13, while L122 and L123 showed single insertions, on Chr02 and Chr06, respectively. Based on the flanking sequences of the inserted T-DNA, the event-specific detection for each event was established using specific PCR primers, and PCR amplification followed by sequencing of PCR products further confirmed the putative insertion loci and flanking regions in the transgenic lines. Our results demonstrate the efficacy and robustness of whole-genome sequencing in identifying the genomic insertions and flanking regions in GM crops. Moreover, the characterization of insertion loci and the establishment of event-specific detection will facilitate the application and development of broad-spectrum virus-resistant transgenic soybean cultivars.

Soybean oil contains approximately 20% oleic acid and 63% polyunsaturated fatty acids, which limits its uses in food products and industrial applications because of its poor oxidative stability. Increasing the oleic acid content in soybean seeds provides improved oxidative stability and is also beneficial to human health. Endoplasmic reticulum-associated delta-12 fatty acid desaturase 2 (FAD2) is the key enzyme responsible for converting oleic acid (18:1) precursors to linoleic acid (18:2) in the lipid biosynthetic pathway. In this study, a 390-bp conserved sequence of GmFAD2-1B was used to trigger a fragment of RNAi-mediated gene knockdown, and a seed-specific promoter of the β-conglycinin alpha subunit gene was employed to downregulate the expression of this gene in soybean seeds to increase the oleic acid content. PCR and Southern blot analysis showed that the T-DNA had inserted into the soybean genome and was stably inherited by the progeny. In addition, the expression analysis indicated that GmFAD2-1B was significantly downregulated in the seeds by RNAi-mediated post-transcription gene knockdown driven by the seed-specific promoter. The oleic acid content significantly increased from 20 to ~ 80% in the transgenic seeds, and the linoleic and linolenic acid content decreased concomitantly in the transgenic lines compared with that in the wild types. The fatty acid profiles also exhibited steady changes in three consecutive generations. However, the total protein and oil contents and agronomic traits of the transgenic lines did not show a significant difference compared with the wild types.