Promoter Analyses and Biochemical Studies of Lettuce (Lactuca sativa) cis-Prenyltransferases

Abstract

Natural rubber is poly-cis-isoprene polymer with molecular weight of more than 1 million Da. Although it is an important raw material used for many industrial and medical products, the biosynthesis of natural rubber is not yet fully understood. Due to the poly-cis-isoprene structure of natural rubber, cis-prenyltransferases are proposed to be responsible for the elongation of this important polymer. Currently, all commercial rubber comes from the Brazilian Rubber Tree (Heava brasiliensis), which is not suited to laboratory study. The annual plant, lettuce (Lactuca sativa), can synthesize high quality NR, is self-pollinating, transformable, and has an available genome sequence. The Ro laboratory at University Calgary, has identified three cis-prenyl transferases (CPTs) and two CPT scaffolding proteins (CSFs) in lettuce. It appears that one pair of a CPT and CSF (LsCPT3 and LsCSF2) is responsible for the production of natural rubber in lettuce, while another pair (LsCPT1 and LsCSF1) is necessary in primary metabolism. The remaining CPT, LsCPT2, is most similar to prokaryotic CPTs and is hypothesized to function without a CSF partner. Currently, there is no specific promoter for natural rubber producing cells (laticifers), which hinders the study of CPT3 and CSF2 in natural rubber production. In this thesis project, promoters for LsCPT3 and LsCSF2 have been cloned and characterized. The GUS reporter gene system was used to demonstrate that both promoters have specific activity in laticifer cells. LsCPT2 was cloned and characterized, demonstrating that LsCPT2 can be expressed in the prokaryotic organism, E. coli. When purified from E. coli, LsCPT2 was found to have CPT activity in vitro, based on a 14C-IPP incorporation assay.