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Molecular biochemical characterization of rubber biosynthetic machinery in Hevea and Dandelion, and evaluation of Rabbitbrush as a potential domestic rubber crop
AuthorHathwaik, Upul I.
AdvisorShintani, David K.
Biochemistry and Molecular Biology
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Natural rubber is an irreplaceable material that is used in manufacturing over 40,000 products including tires and numerous medical devices. Hevea brasiliensis is the sole producer of world natural rubber and more than 90% of the Hevea plantations are located in less than five South Asian countries. In 2011, the US spent 4.4 billion dollars to import natural rubber. When considering the unpredictability and high cost of NR imports, the limited growing conditions of Hevea plants, the increasing allergic reactions caused by Hevea rubber, and the irreplaceability by synthetics, it is clear that domestic sources of natural rubber are needed. Furthermore, the rubber transferase that is responsible for producing rubber is currently unknown, and understanding rubber biosynthetic pathway will help to improve domestic rubber crops. In this study several experiments were done to identify the rubber transferase. The rubber transferase is responsible for the polymerization of isopentenyl pyrophosphate (IPP) monomers into high molecular weight cis-1,4-polyisoprene polymer with the help of farnesyl pyrophosphate (FPP) primer. In all rubber producing plants rubber is made in cytosolic compartments know as rubber particles. The analyses of H. brasiliensis rubber particle proteins with liquid chromatography mass spectrometry (LC/MS) and western blot analysis using cis-prenyltransferase (CPT) antibodies confirmed the CPT localization in washed rubber particles (WRPs). Direct evidence of CPT involvement in rubber polymerization was established by in vitro cross-linking studies done with two different FPP analogues. The in vivo role of CPT was identified by CPT under-expressing transgenic Taraxacum kok-saghyz (Russian Dandelion) plants. ii Additionally, this research was focused in identifying a domestic rubber crop. Ericameria nauseosa (Rabbitbrush) is one of the very few domestic rubber producers. Wild Rabbitbrush populations were analyzed for their rubber amount and molecular weight. Patterns of rubber accumulation and molecular weight in shoots with different diameter tissue were established over time. Rabbitbrush collected from Austin, NV had high quality rubber in high yield. The extracted rubber had several physical properties that were analogous to existing commercial natural rubber producers. The resins and the biomass of Rabbitbrush were also analyzed. Both resin and leftover biomass residue had promising characteristics. Overall, Rabbitbrush collected from Austin, NV has the potential of becoming a domestic natural rubber, resin, and biomass crop.