Mizutani M, Ohta D, Sato R
International Research Laboratories, Ciba-Geigy Japan Ltd., Takarazuka, Japan.
Plant Physiol. 1997 Mar;113(3):755-63. doi: 10.1104/pp.113.3.755.
We have isolated a cDNA for a cytochrome P450, cinnamate 4-hydroxylase (C4H), of Arabidopsis thaliana using a C4H cDNA from mung been as a hybridization probe. The deduced amino acid sequence is 84.7% identical to that of mung bean C4H and therefore was designated CYP73A5. The CYP73A5 protein was expressed in insect cells using the baculovirus expression system and when reconstituted with lipid and NADPH-cytochrome P450 reductase resulted in C4H activity with a specific activity of 68 nmol min-1 nmol-1 P450. Southern blot analysis revealed that CYP73A5 is a single-copy gene in Arabidopsis. C4H (CYP73A5) expression was apparently coordinated in Arabidopsis with both PAL1 and 4CL in response to light and wounding. Although the light induction of CHS followed a time course similar to that observed with C4H, no induction of CHS was detected upon wounding. On the other hand, the C4H expression patterns exhibited no significant coordination with those of PAL2 and PAL3. A C4H promoter region of 907 bp contained all of the three cis-acting elements (boxes P, A, and L) conserved among the PAL and 4CL genes so far reported as controlling expression.
我们以来自绿豆的肉桂酸4-羟化酶（C4H）cDNA作为杂交探针，分离出了拟南芥细胞色素P450——肉桂酸4-羟化酶（C4H）的cDNA。推导的氨基酸序列与绿豆C4H的氨基酸序列有84.7%的同源性，因此被命名为CYP73A5。利用杆状病毒表达系统在昆虫细胞中表达了CYP73A5蛋白，当与脂质和NADPH-细胞色素P450还原酶重组时，产生了具有68 nmol min-1 nmol-1 P450比活性的C4H活性。Southern杂交分析表明，CYP73A5在拟南芥中是单拷贝基因。在拟南芥中，C4H（CYP73A5）的表达显然与PAL1和4CL受光照和创伤诱导的表达相协调。虽然查尔酮合酶（CHS）的光照诱导过程与C4H相似，但创伤处理后未检测到CHS的诱导。另一方面，C4H的表达模式与PAL2和PAL3的表达模式没有明显的协调性。一个907 bp的C4H启动子区域包含了迄今报道的在PAL和4CL基因中保守的所有三个顺式作用元件（P、A和L框），这些元件控制基因表达。
