Database Research Summaries2018 New Breeding Technologies Applied to Meal
Research Focus
The focus of this project is to increase soybean value for the entire value chain, selection of soybean germplasm and identification of soybean genes controlling protein synthesis and storage along with the development of markers for those genes, provide the opportunity to create soybeans with higher protein levels and will aid in the development of commercial soybean varieties with an improved nutritional bundle.
Objectives
- Report on first identification of CRISPR-based knockout mutations in the glycinins/beta-conglycinin gene families (T0 generation).
- Provide target genes for construction of a high sulfur amino acid (methionine or cysteine) construct with seed specific promoters and designed for storage protein body localization.
- Provide transcription factor or other metabolic targets designed to influence protein quantity from analyses of genome and transcriptome data of naturally occurring or transgenic lines.
Results
- The historic protein QTL on chromosome 20 has been targeted for down-regulation in order to better understand the contribution of this gene to the total protein level of seed. We collected T1 seed from 12 transgenic events to date, with an additional 7 events still maturing in the greenhouse. The progeny will be characterized at the molecular level and T2 populations across the respective events monitored for changes in oil and protein levels.
- Using the zygotic embryo culture system, the glycinin promoter was induced by specific amino acids (Gln, Asn, Pro) while no induction was observed with other amino acids (Gly, Met) or other addenda (sorbitol, sucrose, maltose).
- Use of a Cas9-CRISPRi that cannot cut DNA was shown to block gene transcription. Targets near two putative enhancer motifs, as well as the transcription start site in the GmScreamM8 promoter resulted in significant decrease in promoter activity during transient expression.
- CRISPR mutagenesis has also been used to generate knockouts and deletions of the major soybean seed storage proteins. Six different CRISPR constructs have been transformed, two from each of the following categories: (1) Knock out mutations for the four glycinins; (2) Knock out mutations for the nine beta-conglycinins; (3) Deletions for eight of the nine beta-conglycinins. Preliminary evidence indicates that some mutagenesis is occurring in these T0 plants, but it is not yet clear if the mutations will be inherited in the T1 generation.
Importance
This project utilizes gene-editing technology to target families of seed proteins in a stacked approach to meaningfully tip the protein balance in the seed towards storage proteins higher in essential amino acids.
For more information about this research project, please visit the National Soybean Checkoff Research Database.
Funded in part by the soybean checkoff.
