Kater M M, Koningstein G M, Nijkamp H J, Stuitje A R
Department of Genetics, Vrije Universiteit, Amsterdam, Netherlands.
Plant Mol Biol. 1991 Oct;17(4):895-909. doi: 10.1007/BF00037070.
The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastid-localized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific lambda gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.
在油料作物甘蓝型油菜（油菜籽）种子发育过程中，储存脂质生物合成的开始与脂肪酸组成在质量和数量上的急剧变化相一致。在储存脂质生物合成的这个阶段，脂肪酸合酶系统各个组分的酶活性迅速增加。基于其对酰基载体蛋白（ACP）的亲和力，我们描述了一种从发育中的甘蓝型油菜种子中快速、简单地纯化定位于质体的NADH依赖性烯酰-酰基载体蛋白还原酶的方法。对纯化的蛋白质进行N端测序，并用于制备高效抗体。对种子特异性λgt11 cDNA表达文库进行免疫筛选，分离出烯酰-ACP还原酶cDNA克隆。对一个明显全长的cDNA克隆进行DNA序列分析表明，烯酰-ACP还原酶mRNA被翻译成一种前体蛋白，该蛋白具有一个推定的73个氨基酸的前导序列，在蛋白质通过质体膜转运过程中被去除。在大肠杆菌中的表达研究表明，全长cDNA克隆编码真正的甘蓝型油菜NADH依赖性烯酰-ACP还原酶。通过Southern杂交对烯酰-ACP还原酶基因进行表征表明，异源四倍体甘蓝型油菜含有两对相关的烯酰-ACP还原酶基因，它们分别来源于其两个祖先甘蓝和白菜中发现的两个不同基因。对种子发育过程中烯酰-ACP还原酶mRNA稳态水平的Northern印迹分析表明，在储存脂质积累阶段酶活性的增加是在基因表达水平上受到调控的。
