Natural rubber from the para rubber tree Hevea brasiliensis is one of world’s most important natural resources. Despite its use in the manufacture of a wide range of essential items the mechanisms by which natural rubber is synthesised is poorly understood. Natural rubber is a long chain cis-polyisoprene, composed of units of isopentenyl pyrophosphate (IPP) which is contained within rubber particles. Rubber particles consist of a hydrophobic polyisoprene interior surrounded by a monolayer membrane. Due to the insoluble nature of rubber the only place that polymerisation could occur is on this membrane by a hypothetical membrane bound rubber transferase. Whether this is a single enzyme or complex is currently unknown. Cis-prenyltransferases (CPTs) are a group of enzymes responsible for the polymerisation of cis-polyisoprene. The first plant CPTs were identified in Arabidopsis which paved the way for the identification of two Hevea CPTs, HRT1 and HRT2. HRT2 is able catalyse the formation of long chain polyisoprene products in the presence of rubber particles and is the focus of this thesis. Hevea takes at least 4 years to reach maturity. Its long life cycle coupled with the difficulty of genetic transformation meant that direct study on Hevea was not feasible for this project. Instead transient expression in N. benthamiana as well as some preliminary work in L. sativa was used to characterise rubber associated proteins. HRT2 was found to be a cytosolic protein and in theory incapable of polymerising natural rubber on the surface of rubber particles. The scope of the project was therefore widened to include possible interactors that could act to bring it to a membrane. These interactors were identified and cloned based on existing literature and included small rubber particle protein (SRPP), rubber elongation factor (REF), rubber biosynthesis stimulator protein (RBSP) and a newly characterised cis-prenyltransferase like (CPTL) protein, HevNogo. Whilst HRT2 on its own was unable to associate with a membrane, expression with HevNogo induced its subsequent localisation to the plasma membrane. This interaction initially took place on the endoplasmic reticulum. The HRT2/HevNogo complex may be part of a rubber transferase complex, however it is likely that additional components are required.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:714920 |
| Date | January 2016 |
| Creators | Brown, Daniel |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/88568/ |
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