Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720‑3102, USA.
Eurofins Lancaster Laboratories, Professional Scientific Service Division, 2425 New Holland Pike, Lancaster, PA, 17605, USA.
Planta. 2020 Apr 3;251(4):92. doi: 10.1007/s00425-020-03387-1.
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.
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。下调鸟氨酸脱羧酶、精氨酸脱羧酶和天冬氨酸氧化酶导致有活力的植物尼古丁含量显著降低。通过 RNAi 技术降低烟草中的尼古丁积累。该方法下调了编码上游酶的六个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起作用。设计了 9 种不同的 RNAi 构建体来降低编码精氨酸脱羧酶、胍氨酸脱氨酶、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。应用农杆菌介导的烟草叶片转化，经卡那霉素选择，产生 T0 和随后的 T1 代种子。温室中成熟的 T1 植株被剪顶以防止开花，并且在剪顶点以下的第 3 和第 4 片叶子用于检测转录水平和产物积累。精氨酸脱羧酶、天冬氨酸氧化酶和鸟氨酸脱羧酶的下调一致导致相应植物叶片中尼古丁水平降低。具有天冬氨酸氧化酶 RNAi 构建体的转化体在叶片中表现出最低的尼古丁水平，从低于定量下限（20μg/g 干叶重）到 1.3mg/g 干叶重不等。多胺与尼古丁生物合成有关，其主要形式腐胺的含量与精氨酸脱羧酶和鸟氨酸脱羧酶 RNAi 表达转化体中的尼古丁含量呈定性相关。在一些转化株系中观察到衰老表型的假定和较老叶片的存活率降低。这些结果从上述基因在生物碱生物合成途径中的作用角度进行了讨论，并可能有助于指导降低烟草叶片中尼古丁含量的努力。
