Cloning And Functional Analysis Of Soybean High Oleic Acid Related Gene Gm15G117700

Soybean is one of the main source of human edible vegetable oil in the world.The main component of oil are fatty acids,which include palmitic acid,Stearic acid,oleic acid,linoleic acid,linolenic acid and other fatty acids.Among them,oleic acid characterizes soybean oil quality.In fact,oil with high oleic acid content has the advantages of anti-oxidation and storage resistance.Furthermore,soybean oil with a high ratio of oleic acid to linoleic acid（O/L）may prevent cardiovascular and cerebrovascular diseases.Therefore,increasing soybean oleic acid is one of the important goal for soybean breeders.Soybean oleic acid trait as of quantitative nature is controlled by multiple genes.However,except for FAD-2 gene which was known to regulate soybean oleic acid gene expression,the function of other genes is still unknown.In this study,we screened out an important differentially expressed high oleic acid-related gene Gm15G117700 using 260 soybean germplasm resources,a GWAS（Genome-wide association study）population in the preliminary experiment.The gene contributes 15%to the phenotype of oleic acid,but it has not yet been recorded in gen bank,and the function of this gene has not been reported.In this study,we designed specific primers according to the sequence of the gene,and the target gene Gm15G117700 was cloned using PCR technology.The plant expression vector p CAMBIA-3301 was used as the basic vector to construct the overexpression vector p CAMBIA-3301-15G117700.p BGKO53 was used as the basic vector to construct and edit the vector p BGK053-15G117700,which was introduced into the receptor soybean variety’Jinong 38’（JN38）through the agrobacterium-mediated technique and the pollen tube channel method.The preliminary detection of positive transgenic plants was performed by PCR.T₂plants were then subjected to Southern Blotting detection,and fluorescent quantitative PCR was used to identify and detect the fluorescent quantitative expression level of the target gene.A grain analyzer（NIRS DS2500）was used to determine the fatty acid content and analyze the impact of the target gene on the content of oleic acid.The main results were as follows:1.The target gene Gm15G117700 was cloned.The CDS size of the target gene was 693bp.The gene is located on chromosome 15 and encodes a total of 230 amino acids.Bioinformatics analysis predicts that the protein encoded by this gene is a non-secreted protein.The subcellular localization results show that 65.2%was present in the cytoplasm,which mainly plays a role in the cytoplasm.The polypeptide chain as a whole is hydrophilic and belongs to a transmembrane protein.29 potential phosphorylation sites,including 2 tyrosine（Tyr）,12 threonine（Thr）,and 15serine（Ser）phosphorylation sites.The protein spatial structure is dominated by alpha helix and random coils.The gene-encoded protein has 55%of homology with Arabidopsis thaliana in the three-dimensional database.2.Ten（10）soybean genotypes with high and low oleic acid content,respectively were selected for fluorescence quantitative PCR analysis.The results from fluorescence quantitative PCR showed that the Gm15G117700 gene expression level of high oleic acid soybean seeds was lower than 1.50 on average,and the Gm15G117700 gene expression level of low oleic acid soybean seeds was higher than 1.80 on average,suggesting that this gene may play a negative regulatory role in regulating oleic acid synthesis.3.Using the plant expression vector p CAMBIA-3301 as the basic vector,the gene overexpression vector p CAMBIA-3301-15G117700 was constructed with seamless cloning technology.Using p BGKO53 as the basic vector,the CRISPR/Cas9 vector p BGKO53-15G117700 was constructed.The functional elements of the vector mainly include Gm U6promoter,Cas9 protein gene and selection marker Bar.4.Genetic transformation by pollen tube channel method,the overexpression vector and CRISPR/Cas9 vector are transferred into the recipient soybean Jinong 38.After preliminary PCR detection,there were 5 and 4 positive plants in the T₁generation by the pollen tube passage method;10 and 8 positive plants in the T₂generation.Agrobacterium-mediated method transfers the overexpression vector and CRISPR/Cas9 vector into the recipient soybean Jinong 38.After preliminary PCR detection,there were 2 and 3 T₀positive plants by Agrobacterium-mediated method,6 and 7 T₁positive plants,and 7 and 8 T₂positive plants.5.Southern blotting hybridization was performed on the transgenic plants of the T₂generation using the bar gene as a molecular probe.The results showed that the transformed expression elements were integrated into the soybean genome in the form of single copies,with differential signal intensity of hybridization.6.The c DNA samples from roots,stems,leaves,and seeds of T₂plants were used to check the expression levels via fluorescence quantitative PCR.The results showed that the target genes were expressed in the roots,stems,leaves and seeds of the overexpression vector plants at 1.60,1.60,1.32,and 2.40 respectively.The relative expression level was the highest.The expression levels of the target gene in the plant roots,stems,leaves and seeds of the transformation editing vector were 0.40,0.30,0.20,and 0.50,respectively.7.Using the grain analyzer,the preliminary determination of the content of different components of soybean fatty acid in the T₂generation transgenic lines showed that compared with the recipient’Jinong 38’,the stearic acid,palmitic acid,and linolenic acid of the T₂generation transgenic line there was no significant difference in content,and the oleic acid was average increased by 3.49%in p BGK053-15G117700 plants.Linoleic acid average decreased by 5.61%,oleic acid average decreased by 3.94%by p CAMBIA3301-15G117700 plants,and linoleic acid average increased by 1.7%.The suppressed expression of Gm15G117700 gene increased the oleic acid content of soybean seeds after editing,which also indicate that the gene may have a negative regulatory effect on the oleic acid content..8.The results from the agro-morphological characterization of the T₂generation showed that the transgenic Gm15G117700 plants had no obvious changes in plant height,number of nodes,number of branches,umbilical color and other phenotypic traits.