| Polyunsaturated fatty acids (PUFAs) have several important physiological functions in organisms. Linolenic acid, arachidonic acid, and eicosapentaenoic acid are essential PUFAs. To date, PUFAs are mainly isolated from marine fish. However, the origins of PUFAs are insufficient and endangered by the expanding market. Therefore, sustainable and stable sources of PUFAs are urgently needed. Marine microalgae are the primary producers of PUFAs, which are explored as alternative sources without the fish smell and cholesterol content. In this paper, oil-rich micro alga (Isochrysis galbana) is used as experimental material. This algal strain is autotrophic and unicellular, with a total lipid content of up to 44.57% of the dry weight. This strain is also known to accumulate various PUFAs. In addition, the strain is rich in unsaturated fatty acids, which account for more than 50% of the total lipid content. Therefore, the species is ideal material for extracting fatty acid synthetic pathway-related fatty acid desaturases. The oil-rich microalga (I. galbana) has several kinds of fatty acid desaturases and can generate PUFAs. Research on the structure, characteristics, and functions of fatty acid desaturases can reasonably optimize their structures as well as enhance the substrate specificity and enzymatic activity of fatty acid desaturases. This study can provide a theoretical basis for the genetic manipulation of fatty acid desaturases in organisms and the efficient expression of PUFAs. The work and research results of this paper are summarized as follows:1. A total of 18 strains of marine microalgae are screened from the algae library; each strain has a total lipid content of more than 10%. We determined the total lipid content, fatty acid composition, and fatty acid content of 3 Chrysophyta strains,3 dinoflagellate strains,5 Chlorophyta strains, and 7 diatom strains. The total lipid content significantly differed in the 18 strains of marine microalgae. The lipid content of I. galbana is the highest, which accounts for 44.57%±0.54%, followed by Heterosigma sp. (35.2%±0.86%) and Isochrysis zhanjiangensis (29.72%±0.77%).2. The growth, fatty acid composition, and fatty acid content of I. galbana under different culture temperature, salinity, N concentrations, and P concentrations are investigated. Results show that I. galbana has strong environmental tolerance. The microalgae can grow at varied temperature (15-30℃), salinity (15%-60%), N concentration (0-1760μmol/L), and P concentration (0-72.6μmol/L). The optimum growth conditions are a temperature of 20-25℃, salinity of 30%, N concentration of 440-880μmol/L, and P concentration of 18.15-36.3μmol/L. The total lipid content, unsaturated fatty acid composition, and unsaturated fatty acid content significantly differ with environmental factors. Results show that higher temperature and abundant nutrients (N and P) are beneficial to the growth of I. galbana. Low temperature, high salinity, and low N concentration are conducive to the increase of total lipid and unsaturated fatty acid content. The fatty acid composition of I. galbana under different culture conditions is investigated by GC-MS analysis. Results show that several kinds of unsaturated fatty acids are present, including a variety of monounsaturated fatty acids and PUFAs. Therefore, I. galbana is good experimental material for studying the fatty acid desaturase gene family.3. Through transcriptome sequencing of I. galbana, we obtain a large number of gene fragments of fatty acid desaturases. The cDNA of delta-5 and delta-12 fatty acid desaturase genes, IgFAD5 and IgFAD12, respectively, are isolated from I. galbana via RACE method. Sequence analysis indicates that the total length of the IgFAD5 open reading frame (ORF) is 1,320 bp. The deduced amino acid sequence of IgFAD5 is 440 amino acids long, with a predicted molecular weight (MW) of 49.74 kDa and an isoelectric point (pI) of 9.07. The sequence is deposited in GenBank with accession number AFD22890. The IgFAD5 gene is highly conserved and contains the cytochrome b5-like domain (HPGG) and three conserved motifs (HEGGH, HNKHH, and QIEHH). The cDNA of IgFADl2 contains a 1,158 bp ORF that encodes 386 amino acid residues with a deduced MW of 42.80 kDa and pI of 9.20. The sequence is deposited in GenBank with accession number AFB82638. The IgFAD12 gene is highly conserved and contains three conserved motifs (HECGH, HAKHH, and HVVHH). The secondary structures of IgFAD’5 and IgFAD12 include alpha helices, extended strands, and random coils. IgFAD5 and IgFAD12 have four strongly hydrophobic regions, which further indicated that both genes belong to the fatty acid desaturase family.4. The expression levels of IgFAD5 and IgFAD12 are investigated under diverse abiotic stresses, including temperature, salinity, and nitrogen concentration, via reverse transcription polymerase chain reaction. Comparison with growth under the control temperature (20℃), salinity (31%), and original sodium nitrate content of the f/2 medium (880μmol/L), the low temperature (15℃) upregulated the transcription of the IgFAD5 gene at 6-12 h but upregulated the transcription of the IgFAD 12 gene at 6-48 h. High salinity (62% and 93%) upregulated the transcription of IgFAD5 and IgFAD12, respectively. Different N concentrations upregulated the transcription of IgFAD5 and IgFAD12. The expression levels of the two genes are higher under N-limiting (220μmol/L) conditions. Results show significant differences in the expression of IgFAD5 and. IgFAD12 under different environmental conditions.5. To validate the protein activity, the plasmids pYFAD5, pYFAD12, and p416 (control) are transformed into the yeast Saccharomyces cerevisiae K601 strain. The total lipid content of the transformants is analyzed by GC-MS. Results show that two novel peaks, which correspond to the ARA (20:4△5,8,11,14) and EPA (20:5△5,8,11,14,17) methyl ester standards, are present in the transgenic S. cerevisiae K601 expressing the IgFAD5 gene. A novel peak, which corresponds to the LA (18:2△9,12) methyl ester standard, is present in the transgenic S. cerevisiae K601 expressing the IgFAD12 gene. By contrast, these peaks are absent in the control yeast. These findings provide the basis for elucidating the fatty acid metabolic pathway of I. galbana. |
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