Cloning and functional analysis of EpGHQH1 in shikonin production of Echium plantagineum

Shikonins are the main naphthoquinones in the root periderm of Echium plantagineum, as well as the hypocotyl and root hairs of young seedlings. These red pigments can be used as dyes or for medicinal uses since ancient times. Geranylhydroquinone 3″-hydroxylase (GHQH) has recently been identified to catalyze the C-3″ site of geranylhydroquinone to form 3″-hydroxy-geranyl-hydroquinone in shikonin biosynthetic pathway. In this study, we cloned a geranylhydroquinone 3″-hydroxylase candidate gene (EpGHQH1) from E. plantagineum, and further characterized its function in shikonin biosynthesis using the hairy root system by overexpression and RNA interference strategies. The obtained results showed that overexpression of EpGHQH1 increased the accumulation of shikonins, showing 2.1-fold higher in acetylshikonin yield than that of control lines. On the contrary, down-regulation of EpGHQH1 resulted in a decline in shikonin production. Furthermore, the EpGHQH1 expression levels in germinated seedlings showed a high positive relation with shikonin accumulation. These findings demonstrated the function of EpGHQH1 in enhancing shikonin biosynthesis of E. plantagineum, and provided the first example of transgenic hairy root system from E. plantagineum for functional analysis of genes in metabolite biosynthesis. We reported a possible GHQH gene (EpGHQH1), and demonstrated its positive role in shikonin biosynthesis of E. plantagineum with the transgenic hairy root system by overexpression and RNAi strategies.