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

(1)CTP：磷酸胆碱胞苷转移酶(EC2.7.7.15，CCT)是植物细胞合成磷脂酰胆碱的限速酶。利用酵母Cct突变体的互补作用，从甘蓝型油菜根部cdna文库中分离到了4个Cct基因的编码区。推导出的甘蓝型油菜酶的氨基酸序列在中心区与鼠酶和酵母酶相似。虽然从甘蓝型油菜和其他生物中克隆的所有CCT都被预测为结构亲水性的，但用其中一个甘蓝型油菜克隆转化的酵母CCT突变体恢复了微体部分和可溶性部分的CCT活性。这些结果与最近的一种观点一致，即酵母细胞含有一种将酵母CCT靶向到膜上的机制，并可能表明甘蓝型油菜CCT与该机制是相容的。(2)四种甘蓝型油菜CCT的cDNA的可用性将使未来能够更详细地表征该蛋白质。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。这类研究目前正在进行中，涉及在转基因植物中过量生产这种酶，以及评估PC含量的变化与耐寒性的相关性。

(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.