文冠果FAD2的序列与功能分析

Sequence and functional analysis of FAD2 gene from Xanthoceras sorbifolia seeds

脂肪酸脱饱和酶2(fatty acid desaturase 2,FAD2)基因是油脂合成代谢的关键酶基因,其编码的蛋白催化油酸(18∶1)进一步脱饱和形成亚油酸(18∶2)。本研究以富含油酸和亚油酸2种优质不饱和脂肪酸的文冠果种胚为试材,采用简并引物策略结合RACE技术,克隆了文冠果FAD2的c DNA序列。序列分析表明,文冠果FAD2除具有植物FAD2特有的3个组氨酸区和N端的芳香族氨基酸区外,还具有3个N—糖基化位点和多个磷酸化位点。通过农杆菌介导,将文冠果FAD2转入拟南芥fad2突变体中进行表达分析。气相结果表明,拟南芥fad2突变体缺失亚油酸,而转入文冠果FAD2的拟南芥fad2突变体的种子油中产生了亚油酸,这表明所克隆的文冠果FAD2基因编码的酶具有催化油酸(18∶1)脱饱和成为亚油酸(18∶2)的活性。

As a critical enzyme in the lipid biosynthetic pathway, fatty acid desaturase 2 （ FAD2 ） is responsible for the desaturated conversion of oleic acid （ 18：1 ） to linoleic acid （ 18：2 ） . With the combination of degenerate PCR and RACE methods, a full-length cDNA of FAD2 fragment was cloned from the developing Xanthoceras sorbifolia embryos that are rich in linoleic acid and oleic acid. Sequence analysis revealed that X. sorbifolia FAD2 has three N-glycosylation sites and several phosphorylation sites in addition to three conserved histidine motifs and an aromatic amino acid enriched signal YRNKL at the C-terminus. Agrobacterium-mediated X. sorbifolia FAD2 was expressed in Arabidopsis thaliana fad2 mutant to identify its physiological function. The results of gas chromatograph （ GC） analysis indicated that the introduction of X. sorbifolia FAD2 in Arabidopsis fad2 mutant resulted in the generation of linoleic acid, a fatty acid normally not existing in Arabidopsis fad2 mutant. This confirmed that the encoded enzyme by cloned X. sorbifolia FAD2 has the activity to catalyze the desaturated conversion of oleic acid （18：1） to linoleic acid （18：2）.