普通油茶两个Δ-12脂肪酸脱氢酶基因序列特征及表达模式研究

Sequence and Expression Characterization of Two Genes Encoding Δ-12 Fatty Acid Desaturases from Camellia oleifera

[目地]研究普通油茶油脂成分形成的调控机制。[方法]通过转录组测序获得2条普通油茶Δ-12脂肪酸脱氢酶基因序列,分别命名为Cofad6和Cofad2-2,并对这两个基因及其编码蛋白的序列特征进行比较,对其基因表达量与脂肪酸成分含量的相关性进行分析。[结果]Cofad6基因c DNA编码区全长1 347 bp,编码448个氨基酸;Cofad2-2基因c DNA编码区全长1 152 bp,编码383个氨基酸。经比对,Co FAD6蛋白与其余物种FAD6蛋白有65.7%83.68%的氨基酸同源,Co FAD2-2与浙江红花油茶FAD2-2蛋白有99.22%的氨基酸同源,与其余物种的蛋白质78.59%81.72%同源。蛋白质二级结构分析表明,Co FAD6和Co FAD2-2均具有一个脂肪酸去饱和酶结构域,属于脂酰-Co A去饱和酶基因家族;两者均为跨膜蛋白,且Co FAD2-2具有定位于内质网的保守模序。定量PCR检测发现,在普通油茶‘长林4号’无性系未成熟种子中,Cofad6表达量随着种子发育先升高后降低,而Cofad2-2基因随着种子发育表达量逐渐降低,与种子油脂中亚油酸、亚麻酸含量变化趋势呈显著正相关,与油酸含量变化呈显著负相关。[结论]推测Cofad2-2基因是调控普通油茶种子油脂中油酸和亚油酸含量的关键基因之一,该研究为普通油茶油脂改良基因工程育种奠定了基础。

[ Objective] Exploring the fatty acids contents regulation mechanism is needed. [ Method ] Two A-12 fatty acid desaturase gene sequences were got from Camellia oleifera developing seeds by transcriptome sequencing method, which named Cofad6 and Cofad2-2. The traits of the two genes and encoding proteins were compared, and the correlation of gene expression level and fatty acids contents were analyzed. [ Result] Cofad6 has an open reading frame （ORF） of 1 347 bp and encodes a 448 amino acid （AA） protein; Cofad2-2 has an 1 152 bp ORF and en- codes 383 AA protein. According to the alignment results, the CoFAD6 has the 65.7%- 83.68% amino acid se- quence similarity with the FAD6 from other species, CoFAD2-2 amino acid sequence exhibits the highest similarity with C. chekiangoleosa （99.22%）, and has the 78.59% - 81.72% amino acid sequence similarity with FAD2 from other species. CoFAD6 and CoFAD2-2 have a fatty acid desaturase domain both, which means they belong to acyl-CoA desaturase family. CoFAD6 and CoFAD2-2 are transmembrane proteins and the CoFAD2-2 has a microbody C-terminal targeting signal motif. Real-time PCR analysis shows that Cofad6 and Cofad2-2 are both expressed in the developing seed of C. oleifera＇ Changlin No. 4＇. The expression level of Cofad2-2 is on the decline with the develo- ping of seed, the variation trend is similar with that of linoleic acid content and opposite with that of oleic acid con- tent. [ Conclusion]It is indicated that Cofad2-2 gene might be one of the key genes to regulate oil quality in C. oleifera seed. This research provides scientific basis for oil quality improvement and molecular breeding in C. oleifera.