Katavic Vesna, Mietkiewska Elzbieta, Barton Dennis L, Giblin E Michael, Reed Darwin W, Taylor David C
Saskatchewan Wheat Pool Agricultural Research and Development, Saskatoon, Canada.
Eur J Biochem. 2002 Nov;269(22):5625-31. doi: 10.1046/j.1432-1033.2002.03270.x.
Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids.
从高芥酸（HEA）甘蓝型油菜、白菜型油菜和甘蓝中克隆出基因组脂肪酸延长酶1（FAE1），并从低芥酸（LEA）甘蓝型油菜品种Westar中克隆出FAE1，通过PCR进行扩增，并在强半乳糖诱导型启动子的控制下在酵母细胞中表达。正如预期的那样，表达来自HEA芸苔属植物FAE1基因的酵母细胞合成了酵母中通常不存在的超长链单不饱和脂肪酸，而表达来自LEA甘蓝型油菜FAE1基因的酵母细胞的脂肪酸谱与对照酵母样品相同。与已发表的关于不同HEA和LEA甘蓝型油菜品种的研究结果一致，对来自HEA和LEA十字花科植物的FAE1蛋白序列进行比较，发现一个关键的氨基酸差异：HEA FAE1序列第282位的丝氨酸残基在LEA甘蓝型油菜品种Westar中被苯丙氨酸取代。使用定点诱变，将甘蓝型油菜品种Westar的FAE1多肽中的苯丙氨酸282残基替换为丝氨酸残基，将突变基因在酵母中表达，气相色谱分析显示存在超长链单不饱和脂肪酸（VLCMFAs），这表明通过单个氨基酸取代，LEA FAE1酶中的延长酶活性得以恢复。因此，首次发现油菜甘蓝型油菜的低芥酸性状可归因于单个氨基酸取代，该取代阻止了二十碳烯酸和芥酸的生物合成。
