Construction and Analysis of GmFAD2-1A and GmFAD2-2A Soybean Fatty Acid Desaturase Mutants Based on CRISPR/Cas9 Technology

The soybean fatty acid desaturase family is composed of seven genes, but the function of each gene has not been reported. Bioinformatics was used to analyse the structure of genes in this family, as well as the correlation between Δ12-fatty acid desaturase II (FAD2) expression and oleic acid content on different days after flowering of soybean. In the present study, CRISPR/Cas9 technology was used to construct single and double mutant knockout vectors of functional genes in the FAD2 family. Analysis of the molecular biology and expression patterns of genes in the FAD2 family, namely, GmFAD2-1A (Glyma.10G278000) and GmFAD2-2A (Glyma.19G147300), showed that they had little homology with other soybean FAD2 genes, and that their function was slightly changed. Sequencing of the target showed that the editing efficiency of the GmFAD2-1A and GmFAD2-2A genes was 95% and 55.56%, respectively, and that the double mutant editing efficiency was 66.67%. The mutations were divided into two main types, as follows: base deletion and insertion. A near-infrared grain analyser determined the following results: In the T(2) generation, the oleic acid content increased from 17.10% to 73.50%; the linoleic acid content decreased from 62.91% to 12.23%; the protein content increased from 37.69% to 41.16%; in the T(3) generation, the oleic acid content increased from 19.15% to 72.02%; the linoleic acid content decreased from 56.58% to 17.27%. In addition, the protein content increased from 37.52% to 40.58% compared to that of the JN38 control variety.