| Owing to the prominent contradiction of exhausting fossil fuels and growing energy demands, the development and utilization of renewable energy has attracted worldwide attention. People are eager to find a substitute for fossil fuels. However, raw material has been the "bottleneck" which restricts the development of biodiesel. Once people pinned the hope on traditional oil crops, food, animal fats, farming and forestry residues, etc. However, these materials have various kinds of drawbacks. Microalgae can grow in oceans, rivers and lakes, without occupying any plough land. They can quite effectively utilize solar energy for autotrophic photosynthesis and lipid enrichment. Microalgae have been considered as an excellent choice of biodiesel for high growth and mass cultivation.Very long-chain polyunsaturated fatty acids (VLCPUFAs), especially EPA and DHA, are very important for cells and have been found significantly alleviate the symptoms of many diseases. As human cannot synthesize enough VLCPUFAs, dietary supplementation of VLCPUFAs is indispensable. Currently, the principal source of VLCPUFAs is deep-sea fish. However, it is insufficient to an expanding market. To exploit sustainable and continuable sources is urgent need. Many kinds of marine microalgae can synthesize VLCPUFAs with high contents, stable composition of fatty acids, and without unpleasant odor of fish. They are becoming good alternative resources of fish with great development potential.Genus Nannochloropsis is one of class Eustigmatophyceae. Nannochloropsis are rich in EPA, so that they are expected to become the biological resources for EPA. In addition, Nannochloropsis have very high oil content that is more than 30% of dry weight. Many microalgae could obtain high oil contents with reduced biomass at the same time. Nannochloropsis have been proposed as the excellent candidates for fuel production with the optimal combination of biomass productivity and lipid content. In short, they are important applied economic microalgae with deficient molecular information. They deserve intensive investigation. In this study, Nannochloropsis oculata was chosen for study of acyl-coenzyme A synthetase and fatty acid elongase. The cDNA of LACS gene was isolated from N. oculata and named as NOLACS. Nucleotide sequence analysis identified an open reading frame of 1947 bp in length, which encodes a protein of 648 amino acids with a predicted molecular mass of 71 kDa. The predicted amino acid sequence was highly similar to the LACSs of other species,such as Phaeodactylum tricornutum and T. pseudonana. The NOLACS encodes a long-chain acyl-coenzyme A synthetase; it recovered the function of Saccharomyces cerevisiae YB525, a LACS-deficient yeast strain. The substrate specificity of the enzyme was also assayed in the yeast. It was found that NOLACS can activate saturated and some unsaturated fatty acids with a preference to long-chain fatty acids. The effects of nitrogen deficiency on lipid contents and transcription of NOLACS are also assayed.One elongase from N. oculata was cloned by RACE whose ORF is 522 bp encoding 173 amino acids. The results of alignment indicated that NOLACS exhibited higher homology to Phaeodactylum tricornutum, T. pseudonana, Neospora caninum Liverpool etc. The elongase gene was transformed into yeast strain INVSc1(S. cerevisiae) to testify the function. It has been proved that it is a pseudogene.In the light of the great ecological and commercial value, N. oculata deserves intensive investigation at the molecular level. This study not only makes a significant contribution to understanding the metabolic pathways of lipids, but also sets the molecular basis for regulation of gene expression and genetic transformation. The research has important theoretical significance and broad application prospect. |
PDF Full Text Request