Identification of Genes Involved in the Biosynthesis of Tripterygium wilfordii Hook.f. Secondary Metabolites by Suppression Subtractive Hybridization

Abstract

Tripterygium wilfordii Hook.f. (Celastraceae) produces a vast array of natural products, mainly terpenes and sesquiterpene pyridine alkaloids, which have strong biological activities. The limited gene information on the biosynthesis pathways restricts the metabolic engineering of this valuable natural resource. In this study, a suppression subtractive hybridization library of methyl jasmonate (MeJA)-elicited T. wilfordii Hook.f. adventitious root was successfully constructed. To clone the genes involved in the downstream of biosynthetic pathways, a long elicitation time of 12 days was applied. After evaluating the subtraction efficiency through polymerase chain reaction and reverse northern hybridization, 818 clones were randomly sequenced. Basic Local Alignment Search Tool (BLAST) analysis showed that 568 of the 735 successfully expressed sequence tags were annotated. Overrepresentation of gene ontology analysis indicated that the suppression subtractive hybridization library enriched genes that are related to biotic and abiotic stimuli. After BLAST and phylogenetic analyses, 11 genes were found to be putatively involved in the biosynthesis of terpene and sesquiterpene pyridine alkaloid, and nine transcription factors were putatively involved in modulation. The expression modulations of these promising genes were further examined after short MeJA treatment. This study provides valuable information for further research on the production improvement of terpene and sesquiterpene pyridine alkaloids through biotechnology methods.