Guo Yiming, Mietkiewska Elzbieta, Francis Tammy, Katavic Vesna, Brost Jennifer M, Giblin Michael, Barton Dennis L, Taylor David C
National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, SK, Canada S7N 0W9.
Plant Mol Biol. 2009 Mar;69(5):565-75. doi: 10.1007/s11103-008-9439-9. Epub 2008 Dec 11.
Nervonic acid is a Very Long-Chain Monounsaturated Fatty Acid (VLCMFA), 24:1 Delta15 (cis-tetracos-15-enoic acid) found in the seed oils of Lunaria annua, borage, hemp, Acer (Purpleblow maple) and Tropaeolum speciosum (Flame flower). However, of these, only the "money plant" (Lunaria annua L.) has been studied and grown sparingly for future development as a niche crop and the outlook has been disappointing. Therefore, our goal was to isolate and characterize strategic new genes for high nervonic acid production in Brassica oilseed crops. To this end, we have isolated a VLCMFA-utilizing 3-Keto-Acyl-CoA Synthase (KCS; fatty acid elongase; EC 2.3.1.86) gene from Lunaria annua and functionally expressed it in yeast, with the recombinant KCS protein able to catalyze the synthesis of several VLCMFAs, including nervonic acid. Seed-specific expression of the Lunaria KCS in Arabidopsis resulted in a 30-fold increase in nervonic acid proportions in seed oils, compared to the very low quantities found in the wild-type. Similar transgenic experiments using B. carinata as the host resulted in a 7-10 fold increase in seed oil nervonic acid proportions. KCS enzyme activity assays indicated that upon using (14)C-22:1-CoA as substrate, the KCS activity from developing seeds of transgenic B. carinata was 20-30-fold higher than the low erucoyl-elongation activity exhibited by wild type control plants. There was a very good correlation between the Lun KCS transcript intensity and the resultant 22:1-CoA KCS activity in developing seed. The highest nervonic acid level in transgenic B. carinata expressing the Lunaria KCS reached 30%, compared to 2.8% in wild type plant. In addition, the erucic acid proportions in these transgenic lines were considerably lower than that found in native Lunaria oil. These results show the functional utility of the Lunaria KCS in engineering new sources of high nervonate/reduced erucic oils in the Brassicaceae.
神经酸是一种极长链单不饱和脂肪酸（VLCMFA），即24:1 Δ15（顺式二十四碳-15-烯酸），存在于银扇草、琉璃苣、大麻、槭树（紫叶挪威槭）和大花旱金莲（火焰花）的种子油中。然而，其中只有“金钱草”（银扇草）得到了研究，并且作为一种小众作物为未来发展进行的种植规模很小，前景令人失望。因此，我们的目标是分离并鉴定油菜籽作物中用于高产神经酸的关键新基因。为此，我们从银扇草中分离出了一个利用VLCMFA的3-酮酰基辅酶A合酶（KCS；脂肪酸延长酶；EC 2.3.1.86）基因，并在酵母中进行了功能表达，重组KCS蛋白能够催化包括神经酸在内的几种VLCMFA的合成。银扇草KCS在拟南芥中的种子特异性表达导致种子油中神经酸比例增加了30倍，相比之下野生型中的含量极低。以埃塞俄比亚芥为宿主进行的类似转基因实验使种子油中神经酸比例增加了7至10倍。KCS酶活性测定表明，以（14）C-22:1-CoA为底物时，转基因埃塞俄比亚芥发育种子中的KCS活性比野生型对照植物表现出的低芥酸延长活性高20至30倍。在发育种子中，银扇草KCS转录本强度与产生的22:1-CoA KCS活性之间存在非常良好的相关性。表达银扇草KCS的转基因埃塞俄比亚芥中神经酸的最高水平达到30%，而野生型植物中为2.8%。此外，这些转基因品系中的芥酸比例远低于天然银扇草油中的比例。这些结果表明了银扇草KCS在构建十字花科中高神经酸/低芥酸油新来源方面的功能实用性。
