| In recent years, the surge of global fossil fuel consumption caused a worldwide energy crisis. To meet the demands of energy for the economic and social development, to realize the sustainable use of resources, to promote coordinated development of environment, resources, social economy, energy problems attract more and more attention from all over the world. And a cheap green renewable energy has become the goal of world’s governments. Biodiesel, as a main biomass fuels, has become an important substitute for fossil energy due to its low sulfur content, biodegradable, security and high combustion performance, and many other advantages. Marine microalgae has become the hot topics in the study of marine biomass since its high photosynthetic efficiency, strong environment adaptability, short growth cycle and climate without limit, and the advantages of easy to realize large-scale production. Compared to the freshwater microalgae, marine microalgae have a richer biodiversity and higher oil content. The main components of marine microalgae lipid is C14～C18fatty acid triglycerides, which are very suitable for the preparation of biodiesel. In addition, the marine microalgae have the characteristics of no consumption of fresh water resources. This makes marine microalgae potential to prepare biodiesel.Transcription factor is a specific DNA binding protein which could have an interaction with the cis-elements in gene promoter regions and then activate or suppress some gene transcription. Unlike genetic alterations technology which aimed at a single gene, gene transcription factor technology affects a series of metabolic paths. And the metabolic pathways complete the positive or negative control at the same time. The research of transcription factors related in accumulation of oil is great potential in preparation of biodiesel.Polyunsaturated fatty acid is an important substance in organism and also plays an important role on metabolism. Studies have shown that polyunsaturated fatty acids can adjust the ion channels, resist oxidation and can affect swallowing function in cells and the formation of plant chloroplasts. Polyunsaturated fatty acids can also regulate the genetic copy and other important biological processes through the intracellular signal transduction.Nannochloropsis sp. is a kind of micro single-celled algae with a diameter of2to5μm. It is classified into two categories, seawater and freshwater species. Nannochloropsis sp. cells are approximately spherical and live alone or together. Cells are wrapped in a layer of thin cell wall. They contain a pigment and one or three starch grains. Only chlorophyll a is found in Nannochloropsis. Because of its rich in protein, pigment and polyunsaturated fatty acids, Nannochloropsis is widely used in aquaculture. Nannochloropsis is rich in a variety of polyunsaturated fatty acids, especially EPA whose content can reach more than30%of the total fatty acid content.In this study, we cloned fatty acyl coenzyme A synthetase cDNA gene from Nannochloropsis, named it NgLACS. Through the software analysis it was found that the gene was2350bp in length, contained an88bp of the5’-UTR, a312bp of3’-UTR and a1950bp of open reading frame. This gene encoded a protein containing649amino acids. The protein molecular weight is71kDa and the isoelectric point is pH=7.8. The results of TmPred algorithm to predict the transmembrane regions of NgLACS protein showed that three hydrophobic regions located in83-160,103-121,348-368were three transmembrane areas.To clarify the effect of NgLACS on yeast fatty acid composition, we extracted the fatty acids from the Saccharomyces cerevisiae with recombinant plasmid pYLACS and empty pYES2plasmid. The relative content of each fatty acid methyl ester was determined using GCMS. The results revealed that compared to the Saccharomyces cerevisiae with pYES2, the recombinant Saccharomyces cerevisiae had a lower relative content of long chain fatty acids. And some long chain polyunsaturated fatty acids such as C20:2Δ11c,14c, C20:3Δ11c,14c,17c, C20:4Δ5c,8c,11c,14c and C20:5Δ5c,8c,11c,14c,17c, were detected in the recombinant Saccharomyces cerevisiae. In addition, further researches on the effect of different temperature, concentration of nitrogen and Fe3+on the transcription level of NgLACS gene in Nannochloropsis were also performed.In this study, a transcription factor, named NgAUREO1was also cloned in Nannochloropsis. From sequence analysis, the gene had a59%similarity with aureochromel which was found in Vaucheria before. Both of them are constructed by an alkaline leucine zipper (bZIP) in N terminal and a light oxygen voltage-regulated protein (LOV) in C terminal. The ORF encoding477amino acids was129amino acids longer than the aureochromel in Vaucheria. In order to study the relationship between NgAUREO1and fatty acid synthesis, we transformed NgAUREO1gene into Saccharomyces cerevisiae, and expressed it then measured the fatty acid content in the recombinant Saccharomyces cerevisiae. Compared to the yeast with empty plasmid, the oil content in the recombinant yeast was about1.6times more.Gene is the basis of life and the source of metabolism. The study of bioinformatics is the important prerequisite to figure out metabolic mechanism. This study will not only be useful the application of Nannochloropsis, but also provides a new direction of the research on fatty acid metabolism in Nannochloropsis. And also provides a molecular basis for the regulation of metabolism in Nannochloropsis. Therefore, it has a broad prospect of application and an important theoretical significance. |
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