Cazzonelli Christopher I, McCallum Emily J, Lee Rebecca, Botella José Ramón
Department of Botany, Plant Genetic Engineering Laboratory, University of Queensland, Brisbane, Australia.
Transgenic Res. 2005 Dec;14(6):941-67. doi: 10.1007/s11248-005-2539-2.
We report the cloning and characterization in tobacco and Arabidopsis of a Vigna radiata L. (mung bean) promoter that controls the expression of VR-ACS1, an auxin-inducible ACC synthase gene. The VR-ACS1 promoter exhibits a very unusual behavior when studied in plants different from its original host, mung bean. GUS and luciferase in situ assays of transgenic plants containing VR-ACS1 promoter fusions show strong constitutive reporter gene expression throughout tobacco and Arabidopsis development. In vitro quantitative analyses show that transgenic plants harboring VR-ACS1 promoter-reporter constructs have on average 4-6 fold higher protein and activity levels of both reporter genes than plants transformed with comparable CaMV 35S promoter fusions. Similar transcript levels are present in VR-ACS1 and CaMV 35S promoter lines, suggesting that the high levels of gene product observed for the VR-ACS1 promoter are the combined result of transcriptional and translational activation. All tested deletion constructs retaining the core promoter region can drive strong constitutive promoter activity in transgenic plants. This is in contrast to mung bean, where expression of the native VR-ACS1 gene is almost undetectable in plants grown under normal conditions, but is rapidly and highly induced by a variety of stimuli. The constitutive behavior of the VR-ACS1 promoter in heterologous hosts is surprising, suggesting that the control mechanisms active in mung bean are impaired in tobacco and Arabidopsis. The 'aberrant' behavior of the VR-ACS1 promoter is further emphasized by its failure to respond to auxin and cycloheximide in heterologous hosts. VR-ACS1 promoter regulatory mechanisms seem to be different from all previously characterized auxin-inducible promoters.
我们报道了在烟草和拟南芥中对一种绿豆启动子的克隆及特性研究,该启动子可控制生长素诱导型ACC合成酶基因VR - ACS1的表达。当在与其原始宿主绿豆不同的植物中进行研究时,VR - ACS1启动子表现出非常不寻常的行为。对含有VR - ACS1启动子融合体的转基因植物进行GUS和荧光素酶原位分析表明,在烟草和拟南芥的整个发育过程中,报告基因均呈现强烈的组成型表达。体外定量分析表明,携带VR - ACS1启动子 - 报告基因构建体的转基因植物中,两种报告基因的蛋白质和活性水平平均比用可比的CaMV 35S启动子融合体转化的植物高4 - 6倍。VR - ACS1和CaMV 35S启动子系中的转录水平相似,这表明在VR - ACS1启动子中观察到的高水平基因产物是转录和翻译激活的综合结果。所有保留核心启动子区域的测试缺失构建体均可在转基因植物中驱动强烈的组成型启动子活性。这与绿豆形成对比,在绿豆中,正常生长条件下植物中几乎检测不到天然VR - ACS1基因的表达,但在多种刺激下会迅速且高度诱导表达。VR - ACS1启动子在异源宿主中的组成型行为令人惊讶,这表明在绿豆中起作用的控制机制在烟草和拟南芥中受到损害。VR - ACS1启动子未能在异源宿主中对生长素和环己酰亚胺作出反应,这进一步凸显了其“异常”行为。VR - ACS1启动子的调控机制似乎与所有先前表征的生长素诱导型启动子不同。