The role of six alkaloid biosynthesis genes in the process of nicotine accumulation in tobacco was investigated. Downregulation of ornithine decarboxylase, arginine decarboxylase, and aspartate oxidase resulted in viable plants with a significantly lower nicotine content. Attenuation of nicotine accumulation in Nicotiana tabacum was addressed upon the application of RNAi technologies. The approach entailed a downregulation in the expression of six different alkaloid biosynthesis genes encoding upstream enzymes that are thought to function in the pathway of alkaloid and nicotine biosynthesis. Nine different RNAi constructs were designed to lower the expression level of the genes that encode the enzymes arginine decarboxylase, agmatine deiminase, aspartate oxidase, arginase, ornithine decarboxylase, and SAM synthase. Agrobacterium-based transformation of tobacco leaves was applied, and upon kanamycin selection, T0 and subsequently T1 generation seeds were produced. Mature T1 plants in the greenhouse were topped to prevent flowering and leaf nos. 3 and 4 below the topping point were tested for transcript levels and product accumulation. Down-regulation in arginine decarboxylase, aspartate oxidase, and ornithine decarboxylase consistently resulted in lower levels of nicotine in the leaves of the corresponding plants. Transformants with the aspartate oxidase RNAi construct showed the lowest nicotine level in the leaves, which varied from below the limit of quantification (20 μg per g dry leaf weight) to 1.3 mg per g dry leaf weight. The amount of putrescine, the main polyamine related to nicotine biosynthesis, showed a qualitative correlation with the nicotine content in the arginine decarboxylase and ornithine decarboxylase RNAi-expressing transformants. A putative early senescence phenotype and lower viability of the older leaves was observed in some of the transformant lines. The results are discussed in terms of the role of the above-mentioned genes in the alkaloid biosynthetic pathway and may serve to guide efforts to attenuate nicotine content in tobacco leaves.

中文翻译：

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烟草（Nicotiana tabacum）中生物碱和尼古丁含量的遗传衰减


研究了六个生物碱生物合成基因在烟草尼古丁积累过程中的作用。鸟氨酸脱羧酶、精氨酸脱羧酶和天冬氨酸氧化酶的下调导致存活植物的尼古丁含量显着降低。应用 RNAi 技术解决了烟草中尼古丁积累的减弱问题。该方法需要下调编码上游酶的六种不同生物碱生物合成基因的表达，这些酶被认为在生物碱和尼古丁生物合成途径中发挥作用。设计了九种不同的RNAi构建体来降低编码精氨酸脱羧酶、胍丁胺脱亚氨酶、天冬氨酸氧化酶、精氨酸酶、鸟氨酸脱羧酶和SAM合酶的基因的表达水平。应用基于农杆菌的烟叶转化，并在卡那霉素选择后，产生T0代和随后的T1代种子。温室中成熟的T1植物被打顶以防止开花和叶数。测试低于最高点的3和4的转录物水平和产物积累。精氨酸脱羧酶、天冬氨酸氧化酶和鸟氨酸脱羧酶的下调始终导致相应植物叶子中尼古丁的水平较低。具有天冬氨酸氧化酶 RNAi 构建体的转化子在叶子中显示出最低的尼古丁水平，其变化范围从低于定量限（每克干叶重量 20 μg）到每克干叶重量 1.3 mg。腐胺是与尼古丁生物合成相关的主要多胺，其量与表达精氨酸脱羧酶和鸟氨酸脱羧酶RNAi的转化体中的尼古丁含量显示出定性相关性。 在一些转化株系中观察到了推定的早衰表型和老叶的较低活力。研究结果根据上述基因在生物碱生物合成途径中的作用进行了讨论，并可能有助于指导减少烟叶中尼古丁含量的努力。