Studies of expression of genes related to phosphatidylcholine biosynthesis in Brassica napus.

(1)CTP：磷酸胆碱胞苷酰转移酶（EC2.7.7.15，CCT）是植物细胞中磷脂酰胆碱生物合成的限速酶。通过酵母cct突变体的互补，我从甘蓝型油菜根cDNA文库中分离了四个CCT的cDNA。推导的B.napus酶的氨基酸序列在中央结构域类似于大鼠和酵母酶。尽管所有从B.napus和其他生物体克隆的CCT都被预测为结构亲水性的，但用B.napus cDNA克隆之一转化的酵母cct突变体恢复了微粒体部分以及可溶性部分中的CCT活性。这些结果与最近的观点一致，即酵母细胞含有将酵母CCT靶向膜的机制，并且可能表明B.napus CCT与该机制相容。(2)四个B.napus cDNA用于CCT的可用性将允许将来更详细地表征蛋白质。这将包括酶的潜在亚细胞分布，同工酶的生理重要性及其与PC周转的相关性，以及酶的完整三维结构。这些研究目前正在进行中，涉及在转基因植物中过量生产的酶和PC含量的变化与耐冷冻性的相关性的评估。

(1) CTP : phosphocholine cytidylyltransferase (EC2.7.7.15, CCT) is a rate-limiting enzyme in biosynthesis of phosphatidylcholine in plant cells. I have isolated four cDNA for the CCT from a root cDNA library of Brassica napus by complementation in a yeast cct mutant. The deduced amino-acid sequence of the B.napus enzymes resembled rat and yeast enzymes in the central domain. Although all CCT ever cloned from B.napus and other organisms were predicted to be structurally hydrophilic, the yeast cct mutant transformed with one of the B.napus cDNA clones restored the CCT activity in the microsomal fraction as well as in the soluble fraction. These results are consistent with a recent view that yeast cells contained a machinery for targeting the yeast CCT to membranes, and may indicate that the B.napus CCT was compatible with the machinery.(2) The availability of the four B.napus cDNAs for CCT will allow future characterization of the protein in more detail. This would include potential subcellular distribution of the enzyme, physiological importance of the isozymes and its relevance to PC turnover, and the complete three-dimensional structure of the enzyme. Such studies are currently underway involving over-production of the enzyme in transgenic plants and evaluation of the correlation of the changes in PC content with freezing tolerance.