Cloning and molecular characterization of a delta-6 fatty acid desaturase gene from Isochrysis sp CCMM5001

摘要

The cDNA of the delta-6 (Delta 6) fatty acid desaturase gene IsFAD6 was isolated from the marine microalga Isochrysis sp. CCMM5001. Sequence analysis indicated that the total length of IsFAD6 is 2767 bp, and the gene contains a 1407-bp open reading frame, a 406-bp 5' untranslated region (UTR), and a 954-bp 3' UTR with a typical polyA tail. The gene encodes a protein with 469 amino acids, with a molecular weight of 52.9 kDa and an isoelectric point of 8.6. IsFAD6 contains three conserved histidine motifs and a cytochrome b5 domain. Real-time PCR analysis showed that the expression levels of IsFAD6 were significantly upregulated under diverse abiotic stresses, including low temperature (15 and 25 A degrees C), high salinity (62aEuro degrees), and high nitrogen deficiency (220 mu mol L-1 sodium nitrate), compared with those under control conditions (20 A degrees C, 31aEuro degrees salinity, and 880 mu mol L-1 sodium nitrate in the f/2 medium). The IsFAD6 transcript levels minimally increased under high salinity (93aEuro degrees) and excess sodium nitrate (1760 mu mol L-1). Our findings provided an understanding of IsFAD6 responses to temperature, salinity, and nitrate stresses at the transcriptional level. These results indicated that IsFAD6 cloned from Isochrysis sp. CCMM5001 is involved in acclimation to temperature, salt, and nitrogen stresses. The heterologous expression of IsFAD6 in Saccharomyces cerevisiae futher indicated that IsFAD6 could utilize endogenous substrates to synthesize gamma-linolenic acid (18:3(Delta 6,9,12)) and stearidonic acid (18:4(Delta 6,9,12,15)), which are involved in acclimation to adverse conditions.