Creation Of High Oleic Acid Soybean Mutation Plants By CRISPR/Cas9

Soybean?Glycine max L.Merrill?is an important oil and economic crop.Common soybean oil is easily oxidized because it is high in polyunsaturated fatty acids?PUFAs?.As a monounsaturated fatty acid,oleic acid with the oxidative stability contributes significantly to reduce the risk of cardiovascular disease due to the effect on lessening low-density lipoprotein cholesterol in the bloodstream.Therefore,oleic acid content is the essential indicators to evaluate quality of oil in soybean and development of soybean cultivars with new high oleic acid is one of the most important goals in soybean molecular breeding.In the lipid biosynthetic pathway,the enzyme fatty acid desaturase 2?FAD2?is the key enzyme which is responsible for the conversion from oleic acid to linoleic acid in developing soybean seeds.The two microsomal FAD2-1 desaturases FAD2-1A and FAD2-1B were mainly expressed in developing seeds.They were confirmed to play essential roles in controlling the oleic acid content and were used as targets to develop high oleic acid soybean cultivars.In this study,GmFAD2-1A and GmFAD2-1B genes were directionally edited using CRISPR/Cas9 genome editing technology to obtain FAD2-1 knock-out mutants,which could provide new germplasm resources and method for the breeding of high oleic acid.The main results obtained were listed bellow:1.According to the GmFAD2-1A and GmFAD2-1B genes,the three sites of 20 nt guide RNA?gRNA?targeted to the exon of GmFAD2-1A and GmFAD2-1B were designed and transcribed from the AtU3d,AtU3b,and AtU6-1 promoters,respectively,and then the three target sites of gRNA were ligated to the vector pYLCRISPR/Cas9-DB.Then the recombinant plasmid was transformed into two soybean cultivars Huaxia 3?H3?and Dongnong 50?DN50?by Agrobacterium-mediated transformation.We obtained three H3 transgenic soybean plants and three DN50 transgenic soybean plants,respectively.2.The sequences near the editing site were amplified by the PCR method and sequenced from T₀ transgenic soybean plants.The results showed that only GmFAD2-1A gene was edited in T₀ generation.The mutation rates in the T₀ generation from H3 and DN50 both were33%,which the mutation occurred in a single target.3.The agronomic traits such as plant height,main stem number,branching number per plant,leaf shape,flower color,seed coat color,hilum color and growth period were no significant difference between the transgenic soybeans and non-transformed controls.There were also no significant difference in protein and fat content between the transgenic soybeans and the non-transformed controls,indicated that the mutation of GmFAD2-1A gene had no effect on the phenotypes of agronomic traits and the accumulation of total protein and fat in soybean seeds.4.The content of oleic acid in both GmFAD2-1A-H3 mutant and GmFAD2-1A-DN50mutant were significantly higher than that of the control cultivar.The oleic acid content of GmFAD2-1A-H3 mutant was increased from 20%to 23%and the oleic acid content of GmFAD2-1A-DN50 mutant increased from 17.5%to 23%,which were increased 3%and5.5%,respectively.