Cloning And Functional Analysis Of Genes Involved In Oleic Acid Metabolism In Sesame (Sesamum Indicum L.)

Sesame?Sesamum indicum L.?is an important oil crop with high economic value and is rich of unsaturatedfatty acids.Oleic acid and linoleic acid is the main fatty acid composition of sesame seed oil.Stearoyl acyl carrier protein desaturase?SAD?is the only catalytic enzyme for the conversion of stearic acid to oleic acid and it plays an important role in regulating the proportion of saturated fatty acids and unsaturated fatty acids in plant fatty acids.The conversion of oleic acid to linoleic acid in sesame seeds is catalyzed by l-acyl-2-oleoyl-sn-glycero-3-phosphocholine?¹²-desaturase?FAD2?,which determines the ratio of oleic acid and linoleic acid in sesame seed oil.In this study,the SiSAD andSiFAD2A genes of sesame were cloned and analyzed for their expression in different developmental stages of sesame seeds and in different tissues.The SiSAD andSiFAD2Agenes were amplified,sequenced and compared in fifteenaccessions with high oleic acid content and fifteenaccessions with low oleic acid content,respectively.Several SNPs in these two genes were identified.By construction of the transgenic yeast expression vector and plant expression vector,transgenic yeast strains and transgenic Arabidopsis strains were obtained.The expression of the two genes in transgenic materials was analyzed and the gene function was verificated.In this study,the total CDS sequence ofSi SAD andSiFAD2Ageneswere cloned,and the sequence length was as expected.The results of realtime fluorescence quantitative PCR showed that Si SAD andSiFAD2Agenes were expressed in different tissues of different sesame materials,and the expression level of SiSAD andSiFAD2A was highest in sesame seeds.The expression of SiSAD geneincreased slowly in the early stage of developing seed,and increased quickly in 11d and 14d.The expression of Si FAD2A increased in the early stage of developing seedand decreased in the late stage.The highest gene expression level is at 14d.Gene expression ofSiSAD andSiFAD2A were consistent with the accumulation of oleic acid and linoleic acid in sesame.Genesequences ofSiSAD andSiFAD2Ashowed significant polymorphismin different materials.Si SAD gene sequence in sesame high and low oleic acid materials had obvious polymorphism.There weretwo haplotypes and 11 SNPs in SiSAD,resultingamino acid mutations.SixSNPs werenonsense mutationsand two SNPswere mutations between base amino acids.SiFAD2Agene hadthree haplotypes and 10 SNPs.One SNP led hydroxyl amino acids to aliphatic amino acids;One SNP site was base amino acidto base amino acid,others were nonsense mutations.These mutations mightbe related to oleic acid content in sesame seed.Sesame SiSAD andSiFAD2A genes were transformed into Saccharomyces cerevisiae INVSC1 strain.In the three transgenic SiSAD gene yeast strains,the content of oleic acid?C18:1?increased by 4.7%,0.11%and 2.82%,respectively.The content of oleic acid in yeast strain was significantly increasedbyoverexpressed SiSAD gene,indicating that SiSAD could increase the relative content of oleic acid in yeast strain.The content of oleic acid?C18:1?in the transformed SiFAD2A yeast strain decreased by 12.78%,24.50%and 8.55%,respectively,while linoleic acid?C18:2?increased by 232%,538%and441%.The result revealed that Si FAD2A gene can promote the conversion of oleic acid to linoleic acid in yeast strains.Sesame SiSAD andSi FAD2A genes were transformed into Arabidopsis thaliana.Compared with the control,the contents of stearic acid?C18:0?in theSiSAD gene transgenic plants were reduced by 3.0%,3.0%and 6.1%,respectively,andthe oleic acid?C18:1?increased by 2.8%,4.3%and 7.8%,respectively;linoleic acid?C18:2?increased by 4.9%,3.0%and 4.5%,respectively.The ratio of saturated fatty acid to unsaturated fatty acid content?15.8%,15.8%and 15.8%?in transgenic Arabidopsis thalianalower than that in control?16.1%?.The results showed that the overexpression of Si SAD gene could promote the transformation of stearic acid to oleic acid and regulate the ratio of saturated fatty acid and unsaturated fatty acid in plantseeds.Compared with the control strain,linoleic acid?C18:2?increased by 4.42%,9.41%and 4.04%,respectively,inSiFAD2A gene transgenic plants.The results indicated that the SiFAD2A gene could promote the transformation of oleic acid to linoleic acid and regulate the relative content of fatty acids in Arabidopsis thaliana.