甘蓝型油菜烯脂酰-CoA还原酶基因BnECR的克隆及功能分析

doi:10.3724/SP.J.1006.2011.00424

摘要/Abstract

摘要： 反式烯脂酰-CoA还原酶(trans-2, 3-enoyl-CoA reductase, ECR)是催化超长链脂肪酸(VLCFAs)合成的脂肪酰-CoA延长酶之一。根据已报道拟南芥等的ECR基因设计引物，采用RACE (rapid amplification of cDNA ends)方法从甘蓝型油菜中克隆ECR的全长cDNA序列和对应的基因组序列，命名为BnECR (GenBank登录号分别为FJ899705和FJ899706)。序列分析结果显示，BnECR的全长cDNA序列为1 328 bp，对应的基因组序列为2 093 bp，由4个外显子组成，在ORF的上、下游分别有一个163 bp的5′UTR和一个232 bp的3′UTR。根据编码区预测BnECR前体蛋白为一个310个氨基酸残基的多肽链，包含ECR蛋白的重要功能位点K₁₄₄、R₁₄₅及一个NAD(P)H结合基序G₂₂₅SGGYQIPR/HG₂₃₄。NCBI Blastn、氨基酸序列多重比对及保守域分析表明, 该基因与拟南芥AtECR基因的同源性最高，是对应拟南芥AtECR的垂直同源基因。RT-PCR分析表明，BnECR基因在甘蓝型油菜根、茎、叶、花及角果中均有表达，其中在茎中的表达量最高。BnECR在高芥酸材料种子发育中后期的表达量显著高于低芥酸种子，表明BnECR可能参与甘蓝型油菜芥酸的合成。将BnECR克隆到酿酒酵母的穿梭表达载体中，分别转化野生型酵母By4743和突变体菌株YDL015c，添加半乳糖诱导表达。气相色谱分析表明，BnECR在酿酒酵母中有效表达，转化菌株中的芥酸(C22:1)占总脂肪酸含量的1.34%，比对照增加了52%；对突变体的转化结果表明芥酸含量恢复到野生型水平。

关键词: 甘蓝型油菜, 烯脂酰CoA还原酶, 克隆, 芥酸, 酿酒酵母

Abstract: Very-long-chain fatty acids (VLCFAs) are critical components found in cuticular waxes, sphingolipids and triacylglycerols in higher plants. Biosynthesis of VLCFAs is catalyzed by the fatty acyl-CoA elongase, a membrane-bound enzymatic complex containing 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxacyl-CoA dehydratase (HCD), and trans-2, 3-enoyl-CoA reductase (ECR). In this research, primers were designed based on multiple alignments of trans-2,3-enoyl-CoA reductase (ECR) gene sequences from Arabidopsis thaliana and other plants, and the full- length cDNA, here designated BnECR, and the corresponding genomic sequenceswere isolated from Brassica napus by using rapid amplification of cDNA ends (RACE) method (GenBank Accession No. FJ899705 and FJ899705). The sequence of BnECR cDNA was 1 328 bp (excluding the poly dA tail), and the corresponding genomic sequence was 2093 bp. BnECR was composed of four exons and contained a 163 bp 5' untranslated region (5' UTR) and a 233 bp 3' UTR. The deduced BnECR protein was 310 amino acid in length, with a MW of 735.78 kD and a pI of 9.52. The critical functional sites K₁₄₄, R₁₄₅in AtECR were unchanged in BnECR. The G₂₂₅SGGYQIPR/HG₂₃₄which presented a non-classical NADPH-binding motif was found in C-terminal of BnECR. NCBI Blastn, multiple alignments and conserved domain search showed that BnECR had the highest homology to A. thaliana AtECR. RT-PCR analysis showed that BnECR was ubiquitously expressed in B. napus and preferentially expressed in the stem. The transcript level of BnECR at middle and late stages of seed development in low erucic acid rapeseed cultivar was obviously lower than that in high erucic acid rapeseed cultivar, suggesting that BnECR was involved in biosynthesis of erucic acid. The 933 bp BnECRORF was subcloned into the yeast-E. coli shuttle vector pYES2.0. And then the recombinant plasmid was transformed into Saccharomyces cerevisiae wild type strain By4743 and mutant strain YDL015c, respectively. With galactose as inducer, the transformant was cultured to induce the expression of BnECR. The GC result indicated that BnECR was overexpressed effectively in S. cerevisiae, and the content of erucic acid reached to 1.34% of the total fatty acid in recombinant strain, an increase of 52% over the control. Functional complementation of BnECR in a ECR-deficient mutant yeast demonstrated that BnECR mediated the biosynthesis of VLCFAs. Our results suggest that BnECR should be functional orthologue of AtECR.

Key words: Brassica napus L, Enoyl-CoA reductase, Cloning, Erucic acid, Saccharomyces cerevisiae

倪郁, 张飞萃, 王亚超, 蒲飞, 王瑞, 柴友荣, 李加纳. 甘蓝型油菜烯脂酰-CoA还原酶基因BnECR的克隆及功能分析[J]. 作物学报, 2011, 37(03): 424-432.

NI Yu, ZHANG Fei-Cui, WANG E-Chao, BO Fei, WANG Rui, CHAI You-Rong, LI Jia-Na. Cloning and Functional Analysis of Enoyl-CoA Reductase Gene BnECR from Oilseed Rape (Brassica napus L.)[J]. Acta Agron Sin, 2011, 37(03): 424-432.

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