Kisaka Hiroaki, Chin Dong Poh, Miwa Tetsuya, Hirano Hiroto, Uchiyama Sato, Mii Masahiro, Iyo Mayu
Biosolutions Development Section, Biosolutions Labs, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-8681, Japan.
Center for Environment, Health and Field Sciences, Chiba University, 6-2-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
Plant Biotechnol (Tokyo). 2023 Dec 25;40(4):311-320. doi: 10.5511/plantbiotechnology.23.0819a.
The biosynthetic pathway of vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of , then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l kanamycin and 20 mg l meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with , after which genomic PCR analysis using -specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into using an expression vector to join and genes, which are feedback-resistant mutant genes derived from . The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves.
长春花生物碱的生物合成途径有着悠久的研究历史，不仅包括代谢中间体的鉴定，还涉及生物合成成分的细胞间运输和积累机制。长春花生物碱途径始于 strictosidine，它是由色氨酸途径的色胺与类异戊二烯途径的裂环马钱子苷缩合生物合成的。因此，增加前体色氨酸的供应可能会提高长春花生物碱的含量或其代谢中间体的含量。许多关于遗传修饰的报道使用培养细胞或毛状根，但涉及转基因植物生产的报道较少。在本研究中，我们首先研究了稳定生产转基因植物的方法，然后利用该技术改造色氨酸代谢系统，以生产色氨酸含量增加的转基因植物。通过用含有植物表达载体的A13菌株感染播种两周后的子叶，然后用添加了50mg/L卡那霉素和20mg/L美罗培南的1/2 B5培养基进行筛选，获得转化植株。从4200个感染的子叶中获得了68株再生植株，之后使用特异性引物进行基因组PCR分析，证实24株植物中存在该基因，转化率为0.6%。此外，我们使用表达载体将和基因导入，这两个基因是来自的反馈抗性突变基因。所得转化植株的形态与野生型完全相同，尽管色氨酸和生物碱含量显著增加，尤其是叶片中的长春质碱。
