Abstract
BackgroundThe regulation of genes related to lipid metabolism by genetic engineering is an important way to increase the accumulation of lipids in microalgae. DNA binding with one finger (DOF) is a plant-specific transcription factor in higher plants, where it regulates carbon and nitrogen metabolic pathways by regulating key genes involved in these pathways. Overexpression of DOF can increase lipid production in plants; however, it is not clear whether overexpression of DOF can increase lipids in microalgae.ResultsIn this study, we cloned a DOF transcription factor, crDOF, from Chlamydomonas reinhardtii. The sequence of this transcription factor is 1875 bp and encodes a peptide of 624 amino acids with a conserved DOF domain. Overexpression of crDOF in C. reinhardtii significantly increased the intracellular lipid content. The content of total fatty acids in the transgenic algae line Tranc-crDOF-12 was 126.01 μg/mg (dry weight), which was 23.24% higher than that of the wild type. Additionally, the content of unsaturated fatty acids in the transgenic Tranc-crDOF-12 line increased significantly. Fluorescence quantitative PCR analysis showed that in the transgenic line Tranc-crDOF-12, the expression levels of BCC1, FAT1, SQD1, MGD1, DGD1 and PGP1 genes were significantly upregulated, while the expression levels of ACP1, ACS1, CIS1 and SQD2 were downregulated.ConclusionsOur results confirm that crDOF increases intracellular lipids in C. reinhardtii by regulating key genes involved in lipid metabolism. According to these findings, we propose that enhancing the lipid content in microalgae by overexpressing DOF may be achieved in other industrial strains of microalgae and be employed for the industrial production of biodiesel.
Highlights
Third-generation biodiesel technology with microalgae lipids as the raw material has received widespread attention
Lipid droplets of transgenic strains were larger than those of cc849. These results showed that crDOF had a function in lipid synthesis similar to that of soybean DNA binding with one finger (DOF) transcription factor and that overexpression of crDOF in C. reinhardtii could significantly increase the amount of intracellular lipids and the content of unsaturated fatty acids
DOF transcription factors can recognise the AAAG or CTTT motif in the promoter region of their binding genes [25]. We find that these motifs are present in the promoter regions of ten selected genes (BCC1, acyl-carrier protein (ACP1), UDP-sulfoquinovose synthase (SQD1), sulfolipid synthase (SQD2), monogalactosyl diacylglycerol esters (MGD1), digalactosyl diacylglycerol synthase (DGD1), fatty acyl–acyl carrier protein thioesterase (FAT1), citrate synthase (CIS1), acetyl-CoA synthetase (ACS1), and phosphatidyl glycerophosphate synthase (PGP1)) associated with lipid metabolism
Summary
Third-generation biodiesel technology with microalgae lipids as the raw material has received widespread attention. Microalgae are not limited by cultivating land, In recent years, genetic engineering has been widely used to improve the amount of lipids in microalgae [3, 4]. Related studies have focused on the overexpression of key enzymes in fatty acid synthesis [5] or in triacylglyceride synthesis [6] and on knockout or inhibition of enzymes that compete with lipid accumulation [7, 8]. Most of the studies concentrated on single-gene/enzyme regulation. The synthesis and consumption of lipids are a complex metabolic network, rather than a single path. The regulation of genes related to lipid metabolism by genetic engineering is an important way to increase the accumulation of lipids in microalgae. Overexpression of DOF can increase lipid production in plants; it is not clear whether overexpression of DOF can increase lipids in microalgae
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