Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family.

Δ12-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family-GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C-and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T1 generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.

Circular RNAs acting as ceRNAs mediated by miRNAs may be involved in the synthesis of soybean fatty acids.

Soybean oil is composed of fatty acids and glycerol. The content and composition of fatty acids partly determine the quality of soybean seeds. Circular RNAs (circRNAs) are endogenous non-coding RNAs that competitively bind to microRNAs (miRNAs) through miRNA recognition elements, thereby acting as sponges to regulate the expression of target genes. Although circRNAs have been identified previously in soybean, only their expression has been investigated without exploration of the competitive endogenous RNAs (ceRNAs) network of circRNAs-miRNAs-mRNAs. In this study, circRNAs in immature pods of a low linolenic acid soybean Mutant 72' (MT72) and the wild-type control 'Jinong 18' (JN18) were systematically identified and analyzed at 30 and 40 days after flowering using high-throughput sequencing technology. We identified 6377 circRNAs, of which 114 were differentially expressed. Gene ontology and KEGG pathway analyses of targeted mRNAs in the ceRNAs network indicated that the differentially expressed circRNAs may be involved in fatty acid transport, suggesting that circRNAs may play a post-transcriptional regulatory role in soybean oil synthesis. This study provides a foundation for future exploration of the function of circRNAs in soybean and presents novel insights to guide further studies of plant circRNAs.

Exploration of GmDof11-lncRNA13082 Module Regulating Oil Synthesis in Plants.

Soybean (Glycine max [Linn.] Merr.) is an important oilseed crop. Although transcription factors (TFs) can coordinate the expression of mRNA and lncRNA, their coordination in the soybean oil synthesis pathway remains unclear. This study examined the interaction between the TF GmDof11 and lncRNA13082 and found that overexpression of GmDof11 led to an increase in the number of Arabidopsis seeds, thousand seed weight, crude protein, hydrolysis amino acid, and soluble sugar. Additionally, it reduced the triglyceride and starch contents and affected the proportion of fatty acids, increasing the contents of palmitic acid, stearic acid, and linolenic acid. The yeast two-hybrid experiments revealed that GmDof11 interacts with GmBCCP1, GmLEC1b, and GmFAB2 proteins. In the RT-qPCR analysis of transgenic soybean roots, it was found that GmDof11 can activate the production of lncRNA13082 and work in conjunction with lncRNA13082 to oversee oil synthesis and nutrient storage. Our research provides robust theoretical evidence for a comprehensive resolution of TF-lncRNA regulation in the soybean oil synthesis network.

Comparative transcriptomic analysis reveals the molecular mechanism underlying seedling heterosis and its relationship with hybrid contemporary seeds DNA methylation in soybean.

Heterosis is widely used in crop production, but phenotypic dominance and its underlying causes in soybeans, a significant grain and oil crop, remain a crucial yet unexplored issue. Here, the phenotypes and transcriptome profiles of three inbred lines and their resulting F1 seedlings were analyzed. The results suggest that F1 seedlings with superior heterosis in leaf size and biomass exhibited a more extensive recompilation in their transcriptional network and activated a greater number of genes compared to the parental lines. Furthermore, the transcriptional reprogramming observed in the four hybrid combinations was primarily non-additive, with dominant effects being more prevalent. Enrichment analysis of sets of differentially expressed genes, coupled with a weighted gene co-expression network analysis, has shown that the emergence of heterosis in seedlings can be attributed to genes related to circadian rhythms, photosynthesis, and starch synthesis. In addition, we combined DNA methylation data from previous immature seeds and observed similar recompilation patterns between DNA methylation and gene expression. We also found significant correlations between methylation levels of gene region and gene expression levels, as well as the discovery of 12 hub genes that shared or conflicted with their remodeling patterns. This suggests that DNA methylation in contemporary hybrid seeds have an impact on both the F1 seedling phenotype and gene expression to some extent. In conclusion, our study provides valuable insights into the molecular mechanisms of heterosis in soybean seedlings and its practical implications for selecting superior soybean varieties.

Genome-wide identification and analysis of long non-coding RNAs involved in fatty acid biosynthesis in young soybean pods.

Long non-coding RNAs (lncRNAs) are non-coding RNAs of more than 200 nucleotides. To date, the roles of lncRNAs in soybean fatty acid synthesis have not been fully studied. Here, the low-linolenic acid mutant 'MT72' and the wild-type control 'JN18' were used as materials. The lncRNAs in young pods at 30 and 40 days (d) after flowering were systematically identified and analyzed using transcriptome sequencing technology combined with bioinformatics tools. A total of 39,324 lncRNAs and 561 differentially expressed lncRNAs were identified. A lncRNAs-miRNAs-protein-coding genes (mRNAs) network was constructed, and 46 lncRNAs, 46 miRNAs and 137 mRNAs were found to be correlated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 12 targeted mRNAs in the competing endogenous RNA network showed that these lncRNAs may be involved in the biological processes of fatty acid transport, lipid synthesis and cell division. Finally, the expression levels of differentially expressed lncRNAs, miRNAs and mRNAs were verified using qRT-PCR. The expression patterns of most genes were consistent with the sequencing results. In conclusion, new information was provided for the study of fatty acid synthesis by lncRNAs in young soybean pods.