不同超长链脂肪酸延长酶基因FAE1芥酸合成功能的比较

Comparison of Erucic Acid Biosynthesis of the FAE1 Genes Encoding the Very-Long-Chain Fatty Acid Elongase from Different Plant Species

植物芥酸是在以FAE1(Fatty acid elongase 1)编码的β-酮脂酰-CoA合酶为关键限速酶的多酶复合体的催化下合成的,主要以TAG形式储存于种子中,是一种重要的油脂化工原料。不同植物来源的FAE1基因序列差异是导致其芥酸合成能力差异的根本原因。为分离和鉴定芥酸高合成能力的FAE1,本研究采用同源克隆法从油菜、海甘蓝、旱金莲和荷包蛋花等4种植物中克隆获得了12条正常编码的FAE1基因序列,并分别构建表达载体在酵母中进行诱导表达和芥酸含量比较分析。结果表明,源于不同植物的12条FAE1基因cDNA序列一致性介于52.1%~99.9%,氨基酸序列一致性介于49.9%~99.8%,FAE1基因具有明显的种属特性。酵母表达及GC-MS分析结果表明,源于绵油328、海甘蓝和荷包蛋花的8个FAE1基因具有超长链脂肪酸合成能力,其中CaFAE1-3合成芥酸能力最强(4.82%),其次为GjFAE1-1(4.53%),LdFAE1合成芥酸能力最弱(0.29%);CaFAE1-3对C20∶1转化率可达95.39%,在高芥酸育种领域具有极大的应用潜力。另外4个源自阳光80和旱金莲的FAE1基因均不具有芥酸合成功能,因为GyFAE1-2、TmFAE1-1和TmFAE1-2在保守的半胱氨酸或(和)组氨酸位点存在突变,而GyFAE1-1存在1个R395K突变,导致酶活丧失。本研究增进了对FAE1基因结构与功能之间关系的认识,为油菜和海甘蓝的高芥酸育种及芥酸性状基因工程改良提供了科学依据。

In plants,erucic acid is synthesized by the catalysis of a multienzyme complex,in which theβ-Ketoacyl-CoA synthase encoded by FAE1(fatty acid elongase 1)is a key rate limiting factor.Erucic acid is mainly stored in seeds in the form of TAG and is an important oleochemical feedstocks.The FAE1 genes in plants are variable on DNA sequence,thus resulting in differences on capability of erucic acid synthesis.To identify and isolate FAE1 genes with the highest capability on erucic acid synthesis,four plant species including Brassica napus,Crambe abyssinica,Tropaeolum majus and Limnanthes douglasii were used and twelve encoded FAE1sequences were obtained from their genomes by homologous cloning method.Each FAE1 gene was sub-cloned into the yeast expression vector,followed by the analysis of erucic acid content in each recombinant yeast under induction culture conditions.The results showed that the twelve FAE1 genes shared 52.1-99.9%and 49.9-99.8%identity on cDNA and amino acid sequence,respectively,showing species-specific characteristics.By GC-MS analysis of all recombinant yeasts,eight FAE1 genes derived from Mianyou328,Crambe abyssinica and Limnanthes douglasii have been demonstrated with the capability to synthesize very-long-chain fatty acids.CaFAE1-3 had the strongest capability to synthesize erucic acid(4.82%),followed by GjFAE1-1(4.53%),and LdFAE1 that was the weakest one(0.29%).In addition,95.39%of the C20∶1 fatty acids were converted by CaFAE1-3,implying great application potential in high erucic acid breeding.The remaining four genes derived from Yangguang80 and Tropaeolum majus were not detected with the capability on erucic acid synthesis.This is possible because of GyFAE1-2,TmFAE1-1 and TmFAE1-2 that contain mutations in the conserved cysteine or(and)histidine sites,and GyFAE1-1 that contains a R395K mutation resulting in loss of enzyme activity.Collectively,this study represented better understanding of the relationship between the structure and function of FAE1 gene,which has implication in higher erucic acid breeding and genetic engineering improvement on erucic acid trait in rapeseed and Crambe abyssinica.