Tatsumi Kanade, Ichino Takuji, Onishi Noboru, Shimomura Koichiro, Yazaki Kazufumi
Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Uji, Kyoto 611-0011, Japan.
Central Laboratories for Key Technologies, Kirin Holdings Company Limited, 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.
Plant Biotechnol (Tokyo). 2020 Mar 25;37(1):39-46. doi: 10.5511/plantbiotechnology.19.1212a.
, a medicinal plant growing in Asian countries, produces shikonin derivatives that are lipophilic secondary metabolites. These red naphthoquinone pigments are traditionally used as a natural drug and a dye in East Asia. In intact plants, shikonin derivatives are produced in the root epidermal cells and secreted into extracellular spaces. The biosynthetic pathway for shikonin derivatives remains incompletely understood and the secretion mechanisms are largely unknown. Understanding the molecular mechanisms underlying shikonin biosynthesis and transport in cells requires functional analysis of candidate genes using transgenic plants. To date, however, standard transformation methods have not yet been established. This study describes an efficient method for transformation using hairy roots by strain A13, present domestically in Japan. Hairy roots of were generated from explants of the axenic shoots that were infected with strain A13. Integration into the genome was assessed by PCR amplifying a transgene encoding green fluorescent protein (GFP) and by monitoring GFP expression. This method enhanced transformation efficiency 50-70%. Although methods for the systematic stable transformation of plants have not yet been reported, the method described in this study resulted in highly efficient stable transformation using hairy roots. This method enables the functional analysis of genes.
紫草是一种生长在亚洲国家的药用植物，能产生亲脂性次生代谢产物紫草素衍生物。这些红色萘醌色素在东亚传统上被用作天然药物和染料。在完整的植物中，紫草素衍生物在根表皮细胞中产生并分泌到细胞外空间。紫草素衍生物的生物合成途径仍未完全了解，其分泌机制也大多未知。了解紫草素在细胞中生物合成和转运的分子机制需要使用转基因植物对候选基因进行功能分析。然而，迄今为止，尚未建立标准的转化方法。本研究描述了一种利用日本本土存在的A13菌株通过毛状根进行紫草转化的有效方法。紫草的毛状根由无菌芽的外植体产生，这些外植体被A13菌株感染。通过PCR扩增编码绿色荧光蛋白（GFP）的转基因并监测GFP表达来评估其基因组整合情况。该方法将转化效率提高了50 - 70%。虽然尚未报道紫草植物系统稳定转化的方法，但本研究中描述的方法通过毛状根实现了高效稳定转化。该方法能够对紫草基因进行功能分析。
