Molecular cloning and characterization of the secoisolariciresinol dehydrogenase gene involved in podophyllotoxin biosynthetic pathway from Tibet Dysosma.

Abstract

Secoisolariciresinol dehydrogenase catalyzes the conversion of secoisolariciresinol into matairesinol that is a central precursor of antiviral and anti-tumor podophyllotoxin. The full - length cDNA encoding secoisolariciresinol dehydrogenase (designated as DtSD) was cloned and characterized from Tibet Dysosma, Dysosma tsayuensis Ying. The full-length DtSD cDNA was 994 bps containing an 837-bp open reading frame encoding a 278-amino-acid polypeptide with a calculated molecular mass of 29.2 kDa and an isoelectric point of 6.32. Comparative analysis indicated that DtSD was similar with other plant SDs at the level of sequence with the highly conserved catalytic motif, Ser-X13-Tyr-X2-Lys. The homology - based structural modeling showed that DtSD was similar to the SD enzyme from Podophyllum peltatum. Tissue expression pattern analysis indicated that DtSD expressed in root, rhizome, petiole and fruit but at different levels. The highest expression level was found in petiole, and the lowest in fruit. The expression of DtSD could not be detected in leaf and flower. Finally, the podophyllotoxin were detected in all the six tested tissues including root, rhizome, leaf, petiole, flower and fruit at different levels. The highest content of podophyllotoxin (180.5±2.74 μg/g) was found in rhizome, and then followed by root, flower, leaf and petiole. The lowest content of podophyllotoxin (6.03±0.12 μg/g) was found in fruit. The expression levels of DtSD were not consistent with the content of podophyllotoxin in the six tested tissues. So, this might mean that the biosynthetic tissues of podophyllotoxin precursors were not the storage tissues. Key words: Dysosma tsayuensis Ying, secoisolariciresinol dehydrogenase, cloning, expression profile, podophyllotoxin, content.