Effects Of Regulating The Key Genes In Very Long Chainfatty Acid Biosynthesis Pathway On Fatty Acid Composition In Plant Seeds

Very long chain fatty aicds(VLCFAs) are fatty acids with an acyl chain of 18 carbons and longer. The chain length, the degree of unsaturation, the type of polar head and the associated lipids provide the structural and functional diversity of these fatty acids. VLCFAs are imperative to eukaryotes survival and play diverse roles throughout the development. In addition, VLCFAs are important feedstocks for industrial, pharmaceutical and nutraceutical applications. Vegetable oil, as neat fuel, has been to the main source of VLCFAs; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement.This study was aimed to investigate the effects on fatty acid composition in seeds by regulating the key genes in VLCFA biosynthesis pathway, and provide useful information for future engineering of oilseed crops for higher VLCFA production. We regulated the key genes in the biosynthesis pathway of erucic acid and nervonic acid. The results of this study are as follows:1. The Bna A.FAE1 and Bna C.FAE1 were cloned from genome of Brassica napus. There are 21 single nucleotide polymorphisms(SNPs) between the sequence of Bna A.FAE1 and Bna C.FAE1, but only five of them resulted in the difference between amino acids of them. Bna A.FAE1 and Bna C.FAE1 were seperately expressed in seeds of Camelina sativa. The content of C22:1 in seeds from Bna A.FAE1-expressing lines was higher than that in seeds from Bna C.FAE1-expressing lines; whereas the content of C22:0 in seeds from Bna C.FAE1-expressing lines was higher than that in seeds from Bna A.FAE1-expressing lines. All these results suggests that Bna A.FAE1 prefers to use mono-unsaturated fatty acids as substrates, while Bna C.FAE1 inclines to use saturated fatty acids as substrates. However, the Bna A.FAE1 and Bna C.FAE1 were also expressed in fad2/fae1 Arabidopsis thaliana double mutant and Saccharomyces cerevisiae, the difference of substrate specificity between Bna A.FAE1 and Bna C.FAE1 was not reappeared. After analysis of fatty acid composition of Brassica rapa and Brassica olercea which are ancestors of B.napus, the substrate specificity of FAE1 in B.rapa and B.olercea did not show difference.2. B.napus with high oleic/low polyunsaturated fatty acid was crossed with a high erucic acid rape line to introduce the mutated FAD2 and FAD3 into the high erucic acid rape line, and to create the B.napus with high erucic acid/low polyunsaturated fatty acid. In order to avoid bring the mutated FAE1 genes into the high erucic acid rape line, we developed a co-dominant molecular marker associated with Bn FAE1. The Bn FAE1 molecular marker were combined with already developed molecular markers of Bn FAD2 and Bn FAD3, which were used for selection of high erucic acid/low polyunsaturated fatty acid individual plant in F2 population. Two individual plants were selected and fatty acid composition of seeds were detected. The contents of C18:2 in seeds from these two plants were lower than 10%; C18:3 contents were lower than 5%; and C22:1 contents were higher than 40%.3. Bn FAE1 was overexpressed in seeds of B.napus, and the content of C22:1 in seeds was increased from 47% to 60%. Bn FAD2 expression was suppressed by the antisense expressing in seeds of B.napus. The content of C18:2 was decreased from 12% to 7-8%, while the contents of C18:1 and C18:2 were increased. Bn FAE1-expressing line was crossed with antisense of Bn FAD2-expressing line, and some individual plants were selected from F2 population as the erucic content was higher than 60%. The highest content of erucic acid in seeds from selected F2 plants was 63%. Three LPAAT genes, SLC1, SLC1-1 from yeast and Bo LPAT from B.olercea, were expressed in B.napus respectively, but none of them was able to insert erucic acid into sn-2 position of triacylglycerol. Ld LPAAT from Limnanthes douglasii was co-expressed with Bna A.FAE1 in seeds of B.napus, and the content of C22:1 in seeds was significantly increased. The RNAi cassette of Bn LPAAT was co-expressed with Ld LPAAT and Bna A.FAE1 in seeds of B.napus, and the content of C22:1 in seeds was also significantly increased.4. We systematical y investigated the combinatorial effects of KCR, HCD and ECR with KCS on production of VLCFA. La KCS from Lunaria annua was expressed in camelina seeds, and resulted in the accumulation of C24:1. The maximum nervonic acid content in seeds amounted up to 12%. At KCR, At HCD and At ECR from Arabidopsis were separately co-expressed with La KCS in camelina seeds, but nervonic acid content in seeds from coexpressing lines did not show further increases compared with that from the La KCSexpressing line. At KCR and At HCD were co-expressed with La KCS in camelina seeds, and also failed to further increase the content of C24:1. However, C24:1 content from La KCS, At KCR and At HCD co-expressing line was significantly higher than that in La KCSexpressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered.These results suggest that manipulation of the key genes in VLCFA biosynthesis pathway can increase the contents of VLCFAs. This study provide useful information for future engineering of oilseed crops for higher VLCFA production.