| ?-Linolenic acid ( GLA, C18:3?6, 9, 12) is an essential fatty acid, which biosynthesis is catalyzed by ?6-desaturase. Forage is the main source of ruminant nutrition, improving GLA levels in forage has great value to animal health and quality of animal product. Microula sikkimensis, a rare wild oil plant, is known as a new source of GLA. However, to date, the ?6-desaturase gene of M. sikkimensis and the regulation mechanism of the gene by environmental factors have not been reported. In this research,we isolated a gene encoding ?6-desaturase from M.sikkimensis leaves(MsD6DES) and confirmed its function by heterologous expression in tobacco.Meanwhile, the effects of temperature, osmotic stress and wounding stresses on MsD6DES expression and fatty acid composition were analyzed. In addition, other possible role of GLA was also analyzed. This work provided an excellent genetic resource for forage quality improvement, and also obtained a better understanding of the regulatory mechanisms of ?6 fatty acid desaturase gene and GLA by external stimuli. The main results are as follows:(1) The full length of MsD6DES is 1713 bp, the open reading frame is 1,357 bp and encodes 448 amino acids. MsD6DES contained a cytochrome b5-like domain, a heme-binding region, at the N-terminus. The deduced amino acid sequence of MsD6DES showed high similarity (>60%) to other plant A6-desaturases and shared over 80% of similarity with the plants in the same family.(2) MsD6DES expression levels were higher in the seeds and young leaves, and lower in the stems, roots, and mature leaves.(3) Heterologous expression in tobacco indicated that MsD6DES can use endogenous substrates linoleic acid (LA, C18:2?9, 12) and a-linolenic acid (ALA,18:3?9,12,15) to synthesize GLA and octadecatetraenoic acid (OTA, 18:4A6,9,12,15),which account for 1.2% and 1.6% of total fatty acids, respectively. The conversion rates of LA to GLA and of ALA to OTA were 8.4% and 3.4% respectively, which indicate that MsD6DES prefers ?6 substrate LA.(4) Under low temperature stress, unsaturated fatty acids of M. sikkimensis increased, saturated fatty acids content decreased; while opposite trends showed in response to high temperature stress; GLA content increased first and decreased thereafter in both two treatments. Compare to those under high temperature stress, the expression level of MsD6DES gene was higher, and the peak level of MsD6DES expression came earlier in response to low temperature stress. Temperature regulate MsD6DES gene expression at transcriptional level, and the change of GLA content mainly controlled through the transcriptional and post-transcriptional in M.sikkimensis leaves when incubated under high temperature stress.(5) Under osmotic stress, no significant differences of saturated and unsaturated fatty acids content were detected in M. sikkimensis. MsD6DES gene expression was regulated at transcriptional level by osmotic stress stress. There may be a post-transcriptional control of GLA synthesis in M. sikkimensis leaves under osmotic stress.(6) The levels of precursors of jasmonic acid, LA and ALA, increased at 6 h under the wounding treatment, while GLA and ALA contents decreased, which indicated that wounding may induce the pathway of jasmonic acid biosynthesis but inhibit the pathway of fatty acids biosynthesis. MsD6DES was found to be wound-inducible, and GLA also accumulated at 12 h. These results indicated that the MsD6DES and GLA might be involved in the response of M. sikkimensis to wounding stress.(7) GLA?ALA and MeJA are elicitors of an oxidative burst in response to the biotic and abiotic stresses in M. sikkimensis. The activity of proteinase inhibitors could not be changed by GLA, which indicates that GLA is not the precursor of JA. |
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