Genetic Manipulation of Transcriptional Regulators Alters Nicotine Biosynthesis in Tobacco
Genetic Manipulation of Transcriptional Regulators Alters Nicotine Biosynthesis in Tobacco
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DOI：
10.1093/pcp/pcaa036
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发表时间：
2020-06-01
影响因子：
4.9
通讯作者：
Shoji, Tsubasa
中科院分区：
文献类型：
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作者：
Hayashi, Shunya;Watanabe, Mutsumi;Shoji, Tsubasa
有毒的生物碱尼古丁在烟草属植物的根部产生，主要作为一种专门的代谢产物在叶子中积累。烟草根中一对茉莉酸响应性AP 2/ERF家族转录因子NtERF 189和NtERF 199协同上调尼古丁途径中的一系列代谢和转运基因。在这项研究中，我们探讨了操纵这些转录调节因子的表达，以改变烟草中尼古丁的生物合成的潜力。NtERF 189的瞬时过表达导致本氏烟草（Nicotiana benthamiana）和阿拉塔烟草（Nicotiana alata）叶片中生物碱的产生。通过共过表达编码碱性螺旋-环-螺旋-家族MYC 2转录因子的基因，进一步增强了这种异位生产。NtERF 189的组成型和叶特异性过表达增加了转基因烟草植物中叶生物碱的积累，但对植物生长产生负面影响。相比之下，在通过基于CRISPR/Cas9的基因组编辑获得的NtERF 189和NtFRF 199的敲除突变体中，生物碱水平大幅降低，而不会导致主要的生长缺陷。代谢物分析揭示了操纵尼古丁途径对广泛的含氮和含碳代谢物的影响。我们的研究结果为通过靶向转录因子来工程化代谢途径的生物技术应用提供了见解。
The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtFRF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotech-nological application of engineering metabolic pathways by targeting transcription factors.