Abstract
Lithospermum erythrorhizon produces red naphthoquinone pigments called shikonin derivatives in the roots. In the cell cultures of L. erythrorhizon, stimulators of shikonin biosynthesis (Cu2+ and MJ) as well as its inhibitors (NH4+ and light) have been identified. Shikonin is biosynthesized from geranyldiphosphate derived via mevalonate pathway and p-hydroxybenzoate via shikimate pathway, whereas the key step of naphthalene ring formation is not clarified yet.
To isolate cDNAs specifically expressed under shikonin production, we applied PCR-select cDNA subtraction technique, where dark-inducible genes were enriched. Out of 240 clones whose expressions were enhanced in the dark, we have selected a polyphenol oxidase-like protein as a candidate gene for the naphthalene-forming enzyme. Time course experiment of the northern blot showed that the gene expression completely paralleled the shikonin production pattern by various regulatory factors. We are preparing the recombinant enzyme to characterize its catalytic function.
