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首页> 外文期刊>Plant Physiology and Biochemistry >RNAi based simultaneous silencing of all forms of light-dependent NADPH: Protochlorophyllide oxidoreductase genes result in the accumulation of protochlorophyllide in tobacco (Nicotiana tabacum)
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RNAi based simultaneous silencing of all forms of light-dependent NADPH: Protochlorophyllide oxidoreductase genes result in the accumulation of protochlorophyllide in tobacco (Nicotiana tabacum)

机译：基于RNAi的所有形式的光依赖性NADPH的同时沉默：叶绿素原原氧化还原酶基因导致烟草中原叶绿素的积累

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摘要

Conversion of protochlorophyllide (Pchlide) into chlorophyllide (Chlide), a key step in chlorophyll biosynthesis, is mediated by a light-dependent NADPH:protochlorophyllide oxidoreductase (POR). POR exists in multiple isoforms that share high degree of homology. RNAi-mediated gene silencing approach was used to suppress the expression of POR genes in order to study its role in the Chls biosynthesis in tobacco (Nicotiana tabacum L.). The transgenic plants were devoid of chlorophyll pigments and resembled albino plants. Northern blot analysis confirmed the degradation of POR transcripts into 21-23bp fragments. Pigment analysis showed the accumulation of various intermediate compounds of Chl biosynthesis pathway including Pchlide. However, no trace of chlorophyll was observed. As compared to wild type, POR-silenced plants accumulated larger (60%) amounts of Pchlide from its endogenous substrate. When leaf discs of WT and POR-silenced plants were treated with exogenous ALA both WT and POR-silenced plants accumulated large amounts of tetrapyrrolic intermediates demonstrating that Pchlide biosynthesis potential was not suppressed in POR-silenced plants. Upon illumination, WT plants photo-transformed large amounts of Pchlide to Chlide. However, POR-silenced plants almost completely failed to do so. Results demonstrate that the antisense approaches to drop expression of individual POR isoforms have provided valuable insights into the role of distinct PORs during greening. Moreover, data illustrate that the POR is the only enzyme that can convert the Pchlide to Chlide and there is no alternate enzyme that can substitute the POR in higher plants. Thus, this investigation describes ideal mechanism for the silencing of POR isozymes in tobacco.
机译：叶绿素生物合成的关键步骤是将原叶绿素（Pchlide）转化为叶绿素（Chlide），这是由光依赖性NADPH：原叶绿素氧化还原酶（POR）介导的。 POR存在多个具有高度同源性的同工型。为了研究POR基因在烟草（Nicotiana tabacum L.）的Chls生物合成中的作用，使用了RNAi介导的基因沉默方法来抑制POR基因的表达。转基因植物不含叶绿素色素，与白化植物相似。 Northern印迹分析证实了POR转录物降解为21-23bp的片段。颜料分析表明，包括Pchlide在内的Chl生物合成途径的各种中间化合物的积累。但是，没有观察到叶绿素的痕迹。与野生型相比，POR沉默的植物从其内源性基质中积累了更多（60％）数量的苯胺。当用外源ALA处理野生型和POR沉默的植物的叶盘时，野生型和POR沉默的植物均积累了大量的四吡咯中间体，这表明在沉默后的POR中未抑制Pchlide的生物合成潜力。照亮后，野生植物将大量的Pchlide光转化为Chlide。然而，POR沉默的植物几乎完全没有这样做。结果表明，降低单个POR亚型表达的反义方法已为了解绿色中不同POR的作用提供了有价值的见解。此外，数据表明，POR是唯一可以将Pchlide转化为Chlide的酶，在高等植物中没有替代酶可以替代POR。因此，本研究描述了使烟草中POR同工酶沉默的理想机制。

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