Establishment of Rhizobium-mediated transformation of Coptis japonica and molecular analyses of transgenic plants

Abstract

Coptis japonica is a perennial medicinal plant grown in Asia, and its rhizome is used as crude drug, in which berberine is highly accumulated as the main alkaloid. In this study, a transformation method for C. japonica using Rhizobium has been established. Young petioles were infected with Rhizobium radiobactor Gv3101 (pMP90) harboring a plasmid with β-glucuronidase (gus) driven by cauliflower mosaic virus 35S promoter and hygromycin phosphotransferase (hpt) gene as the selection marker. GUS assay showed that 46% of the calli grown on hygromycin plates clearly expressed the gus gene. As an application of the transformation method, an endogenous cDNA encoding an ABC protein, CjMDR1, which is involved in berberine transport, was introduced in sense orientation. Cjmdr1 transgenic plant (Sense-2) regenerated from hygromycin resistant callus line had a single copy of integrated hpt gene in its genomic DNA. Northern analyses of the Sense-2 plant showed that Cjmdr1 mRNA levels were lower in all organs examined than those of wild-type plant, which suggested that co-suppression of Cjmdr1 expression occurred. Indeed, the berberine content in Sense-2 was also suppressed. This is the first report of stable transformation of Coptis spp. and also of the alteration of a secondary metabolite by transport engineering.