CDNA Cloning And Overexpressing Hairy Root Induction Of LeHMGR Gene In Lithospermum Erythrorhizon

Shikonin and its derivatives are a class of naphthoquinone compounds only synthesized in the roots of Boraginaceae plants, such as Arnebia euchroma(Royle) Johnst, Onosma paniculatum Bur. et Franch and Lithospermum erythorhizon sieb. et zucc. These compounds possess significant anti-microbial, anti-inflammatory, anti-tumor, anti-HIV and other important medicinal activities. Up to now, the biochemistry and enzymology of shikonin biosynthesis pathway are well understood, and many key enzymatic genes have been cloned and characterized. However, since the shikonin biosynthesis was regulated by many kinds of impact factors, such as light signals, plant hormones, inorganic elements, fungal elicitors and so on, the molecular regulatory mechanism of shikonin biosynthesis was still largely unknown.Shikonin and its derivatives are biosynthetically derived from two key precursors, p-hydroxybenzoic acid (PHB), derived from phenylpropanoid metabolites, and geranyl pyrophosphate (GPP), formed from themevalonate acid (MVA) pathway. Then these two key precursors are catalyzed by PGT and formed m-geranyl-p-hydroxybenzoic acid (GBA), by a series of metabolic cascade enzymes formed in the endoplasmic reticulum, and through the extracellular secretion transported the shikonin microparticles to the cell wall outside the epidermal cells. One important metabolic intermediate is formed from the mevalonate acid (MVA) pathway, in which HMGR is the first rate-limiting enzyme. Therefore, cloning and inducing the HMGR-overexpressing hairy roots of Boraginaceae plants has important theoretical and practical value.In this study, we successfully cloned the full-length cDNA sequence of LeHMGR gene by using the Rapid Amplification of cDNA Ends (RACE) method based on the partial core sequence obtained with the transcriptome sequencing of L. erythrohizonsin our laboratory. Then, the plant overexpression vector LeHMGR-PBI121-GFP was constructed and transformed into Agrobacterium rhizogenesstrainATCC15834. Finally, LeH7MGR-overexpressing (OH) and wild type (WT) hairy roots were induced separately by infecting the aseptic explants of L. erythrorhizon seedlings. Subcellular localization analysis showed that the gene is possibly localized in the cell wall or plasma membrane, which needs further study. These results provide experimental materials for the study of regulation and molecular mechanism of LeHMGR in shikonin and its derivatives biosynthesis.