红花ω-3脂肪酸脱氢酶基因FAD3的克隆及表达分析

Cloning and Expression Analysis of ω-3 Fatty Acid Dehydrogenase Gene FAD3 from Carthamus tinctorius L.

亚麻酸属植物中的多不饱和脂肪酸,其由微粒体和质体类两种不同类型的ω-3脂肪酸脱氢酶催化合成的,一般认为微粒体ω-3型脂肪酸脱氢酶基因(FAD3)是控制种子油中α-亚麻酸合成的关键酶基因,尽管目前从红花中克隆到了该基因,但在红花种子中未检测到亚麻酸,本研究旨在探究红花种子中不含亚麻酸的原因。利用红花种子转录组数据,根据FAD3基因的开放阅读框两端设计引物,分别以DNA和cDNA为模板进行克隆,并对其进行生物信息学分析、表达水平分析及功能分析。分析结果表明FAD3基因开放阅读框区不含内含子,FAD3氨基酸序列不存在信号肽,其第三个组氨酸保守区为HVVHH,而其他大多数植物均为HVIHH。FAD3蛋白相对保守,富含亮氨酸,存在3个组氨酸保守区。对CtFAD3氨基酸序列317位的缬氨酸替换成异亮氨酸后,发现FAD3基因仍不具有亚油酸脱氢酶活性,且红花中ω-3脂肪酸脱氢酶基因整体上在发育的种子中表达水平是极低的。可推测CtFAD3基因在种子中的低水平表达及其功能的丧失是导致红花种子中不含有α-亚麻酸的重要原因之一。

Linolenic acid is polyunsaturated fatty acid in plants and it is synthesized by two different sorts of ω-3 fatty acid dehydrogenases. It is acknowledged that FAD3 gene is the key enzyme gene controlling α-linolenic acid synthesis in seed oil. Although it has been cloned from safflower successfully, linolenic acid was not detected in safflower seeds. The purpose of our study is to elaborate the phenomenon that linolenic acid is not contained in safflower seeds. Based on the transcriptome data of safflower seeds, primers were designed according to the two ends of FAD3 gene open reading frame, and DNA and cDNA were cloned respectively. Moreover, the bioinformatics analysis, expression level and function analysis of CtFAD3 gene were executed. The results indicated that intron sequence was not included in CtFAD3 gene sequence. There was no any signal peptide in CtFAD3 amino acid sequence. The third histidine conserved region is HVVHH, while most of plants are HVIHH. CtFAD3 protein is much conservative and is rich in leucine. Three histidine conserved regions exist in CtFAD3 protein sequence.Although substituting isoleucine with valine at 317position, CtFAD3 still cannot convert linoleic acid to linolenic acid. Expression level of FAD3 gene in safflower was extremely low in developing seeds. The lower expression level of CtFAD3 gene in seeds and the loss of function may be the critical reasons for absence of α-linolenic acid in safflower seeds.