十字花科植物中低芥酸野生种的发掘和FAE1基因的功能验证

 【目的】发掘低芥酸野生种,并探明这些野生种的低芥酸遗传机理。【方法】本研究采用同源序列法从野生种和甘蓝型油菜中分离FAE1序列。用酵母系统异源表达野生种的FAE1。用气相色谱法分析脂肪酸组成。【结果】比较FAE1序列发现5个野生种的FAE1序列与甘蓝型油菜的FAE1同源性高于85%。分析它们编码的蛋白质序列,发现5个野生种在第282位氨基酸残基处都是丝氨酸,而且与酶活性相关的氨基酸残基（Cys223、His302、His387、His391和His420）都没有发生突变,这些特征与报道的高芥酸油菜品种相同,不同于低芥酸油菜。Western Blot分析发现野生种的FAE1都在酵母系统中获得了表达,转化诸葛菜和荠菜FAE1的酵母并未合成长链脂肪酸,而转化新疆野芥、新疆白芥和菘蓝的FAE1的酵母都有微量长链脂肪酸的合成。【结论】诸葛菜和芥菜确为低芥酸野生种,而且其低芥酸性状源于FAE1编码产物的失活。

Discovery of Low Erucic Acid Wild Species and Functional Characterization of FAE1 Genes in Crucifer Species

[Objective] This study was designed to discover the LEA (low erucic acid) wild species, and to reveal the genetic mechanism of the LEA wait. [Method] Homologous sequence amplification strategy was used in gene isolation. Heterologous expression of the FAE1 genes was made using the yeast system, and the fatty acid composition was analyzed by gas chromatography. [Result] The multiple sequence alignment showed that FAE1 genes shared over 85% identities between the wild species and the rapeseed variety. The amino acid residues (Ser282, Cys223, His302, His387, His391, and His420), reportedly active sites for β-ketoacyl-CoA synthase, were conserved as those in high erucic acid rapeseed. Western blot analysis showed that the FAE1 genes from all of the wild crucifer species were expressed in yeast, whereas the yeast cells expressing FAE1 genes from Orychophragmus violaceus and Capsella buraspastroil showed no formation of very long chain fatty acid (VLCFA). Meanwhile, those expressing FAE1 genes from Sinapis arvensis, Sinapis alba, and Isatis indigotica produced small quantity of VLCFA which was absent in control yeast cells. [Conclusion] The experiment indicated that O. violaceus and C. buraspastroil are LEA wild species in which loss of activity of the protein encoded by the FAE1 gene might be responsible for the LEA trait.