Functional Analysis of Sesame Diacylglycerol Acyltransferase and Phospholipid: Diacylglycerol Acyltransferase Genes Using In Silico and In Vitro Approaches

Sesamum indicum L. is one of the major oilseed crops of India. Sesame oil is stable with a good source of unsaturated fatty acids. To know the mechanism of oil accumulation in sesame, genes coding for the key enzymes involved in triacylglycerol (TAG) synthesis pathway, namely diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) and phospholipid:diacylglycerol acyltransferase (PDAT; EC 2.3.1.158), were retrieved from sesame full genome sequences by comparative analysis using Arabidopsis genes. All isoforms of SiDGAT and SiPDAT genes were analyzed in silico for their sequence and structural similarity with the existing members from other plants. Phylogenetic analysis delineated the sesame DGAT and PDAT genes into four separate clades. Most of the members had several transmembrane domains except SiDGAT1 A1, SiDGAT1 A2, SiDGAT1 B1, and SiPDAT2 which do not have any transmembrane domain suggesting a soluble function. In vitro gene isolation followed by mRNA expression analysis of SiDGAT1, SiDGAT2, SiPDAT1, and SiPDAT2 were carried out. Expression of these genes at the transcription level at the leaf, stem, root, flower, developing seeds, and mature seeds were examined by quantitative real-time PCR experiments. Functional analysis of these sesame genes was performed in a yeast quadruple mutant lacking TAG synthesis genes. Transcript abundance of SiDGAT1 and SiDGAT2 genes was highest in mature seeds, SiPDAT1 at flowering stage and developing seeds, and SiPDAT2 in stem and developing seeds. Heterologous expression of sesame genes in the yeast system indicated higher oil content in both SiDGAT and SiPDAT gene–transformed mutants. In addition, SiPDAT1-expressing mutants had higher polyunsaturated (C18:1; C18:2) fatty acid content. The present study indicates both sesame DGAT and PDAT genes are good candidates for not only oil yield increase but also for higher PUFA content.