Kis Mihaly, Burbridge Emma, Brock Ian W, Heggie Laura, Dix Philip J, Kavanagh Tony A
Institute of Bioengineering and Agroecology, Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
Ann Bot. 2004 Mar;93(3):303-10. doi: 10.1093/aob/mch045. Epub 2004 Jan 28.
Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence.
Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques.
Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels.
There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development.
天然辣根（Armoracia rusticana）过氧化物酶，即辣根过氧化物酶（HRP，EC 1.11.1.7）同工酶C在合成时带有N端和C端肽段延伸，据信这与蛋白质靶向定位有关。本研究旨在探究这些延伸段的具体功能，并培育出具有适合探究过氧化物酶在植物发育和防御中作用的表达模式的转基因植物。
构建了表达合成辣根过氧化物酶同工酶C基因不同版本的转基因烟草植株。该基因经过工程改造，包含编码天然N端或C端延伸段或两者的额外序列。这些构建体置于组成型启动子（CaMV - 35S）或烟草核酮糖-1,5-二磷酸羧化酶小亚基光诱导启动子（SSU）的控制下，并通过农杆菌介导的转化方法导入烟草。为研究N端和C端延伸段的作用，利用生化和分子技术确定重组过氧化物酶的定位。
与野生型烟草水平相比，转基因烟草植株的过氧化物酶活性可提高十倍，且大部分活性位于共质体中。N端延伸段对于高水平重组蛋白的产生至关重要，而C端延伸段影响较小。酶活性水平和重组蛋白水平的差异反映在转录本水平上。
在这个异源表达系统中，没有证据支持重组过氧化物酶存在优先分泌或液泡靶向定位。这使我们对这些肽段延伸段假定的靶向作用产生质疑。N端延伸段对于高水平表达至关重要，似乎影响转录本稳定性或翻译效率。已培育出胞质过氧化物酶活性大幅提高、质外体活性略有增加的植株。这些植株对于探究这些酶在缓解胁迫和植物发育中的作用具有重要价值。
