Two isoforms of NADPH:cytochrome P450 reductase in Arabidopsis thaliana -: Gene structure, heterologous expression in insect cells, and differential regulation
Two isoforms of NADPH:cytochrome P450 reductase in Arabidopsis thaliana -: Gene structure, heterologous expression in insect cells, and differential regulation
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DOI：
10.1104/pp.116.1.357
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发表时间：
1998-01-01
期刊：
影响因子：
7.4
通讯作者：
Ohta, D
中科院分区：
文献类型：
--
作者：
Mizutani, M;Ohta, D
我们已经调查了两个NADPH-细胞色素（Cyt）P450还原酶异构体编码的不同基因（ARI和AR 2）在拟南芥。我们利用绿豆（Phaseolus aureus L.）以NADPH-Cyt P450还原酶cDNA为探针。使用杆状病毒表达系统产生的重组AR 1和AR 2蛋白分别对Cyt c和NADPH显示出相似的Km值。在重组肉桂酸4-羟化酶（CYP 73 A5）的重组系统中，重组AR 1和AR 2蛋白产生相同水平的肉桂酸4-羟化酶活性（约70 nmol min（-1）nmol（-1）P450）。AR 2基因表达瞬时诱导4-和3-倍1小时内的创伤和光处理，分别和诱导时间过程之前的CYP 73 A5和苯丙氨酸解氨酶（帕利）基因。相反，ARI表达水平在处理期间没有变化。AR 1和AR 2基因结构的分析表明，只有AR 2启动子包含三个假定的序列基序（框P，A和L），这是参与协调表达的CYP 73 A5和其他苯丙烷途径基因。这些结果表明，AR 2转录可能在功能上与苯丙素途径酶的诱导水平有关。
We have investigated two NADPH-cytochrome (Cyt) P450 reductase isoforms encoded by separate genes (ARI and AR2) in Arabidopsis thaliana. We isolated AR1 and AR2 cDNAs using a mung bean (Phaseolus aureus L.) NADPH-Cyt P450 reductase cDNA as a probe. The recombinant AR1 and AR2 proteins produced using a baculovirus expression system showed similar K-m values for Cyt c and NADPH, respectively. In the reconstitution system with a recombinant cinnamate 4-hydroxylase (CYP73A5), the recombinant AR1 and AR2 proteins gave the same level of cinnamate 4-hydroxylase activity (about 70 nmol min(-1) nmol(-1) P450). The AR2 gene expression was transiently induced by 4- and 3-fold within 1 h of wounding and light treatments, respectively, and the induction time course preceded those of CYP73A5 and a phenylalanine ammonia-lyase (PALI) gene. On the contrary, the ARI expression level did not change during the treatments. Analysis of the AR1 and AR2 gene structure revealed that only the AR2 promoter contained three putative sequence motifs (boxes P, A, and L), which are involved in the coordinated expression of CYP73A5 and other phenylpropanoid pathway genes. These results suggest the possibility that AR2 transcription may be functionally linked to the induced levels of phenylpropanoid pathway enzymes.