Molecular characterisation of Jatropha Curcas : towards an understanding of its potential as a non-edible oilseed-based source of biodiesel

PhD ThesisJatropha curcas, a non-edible oilseed, is becoming popular as a source of biodiesel. However, there are some limitations to using J. curcas such as the presence of carcinogenic substances, unpredictable yield, asynchronous flowering and plant height. Understanding the genetic control of these characteristics will help to find solutions. To accumulate genetic information for J. curcas plant breeding programmes, the genetic and phenotypic characterisation of J. curcas seeds obtained from different geographical locations was undertaken (Chapter 2). The results showed a narrow genetic diversity among accessions but apparent phenetic diversity. The screens employed ranged from phenotypic characterizations of seeds to molecular, biochemical, physiological and genotyping assessments. Seed dimensions and weight measurements provided data on seed density and kernel weight, both important parameters for assessing oilseed quality. Biochemical analysis provided data on oil properties relevant to biodiesel e.g. total oil content, oleic/linoleic acid ratio, saponification number, cetane number and gross energy value. Expression analysis of genes (acc, sad, fad, and dgat) essential for the quantity and quality of seed/oil were targeted as molecular screens. Understanding the molecular mechanism of lipid cycling in J. curcas seeds will guide future improvements in oil yield. However, this is still an area of active research because little information exists for this in J. curcas. With regards to oil storage capacity of seeds, no information previously existed on the biology of oil bodies in Jatropha curcas. In this study, oil bodies from J. curcas were studied at the gene transcript and protein levels (Chapter 3). Three Jatropha oleosin genes were isolated. Sequence analysis showed that all three oleosin genes contained the proline knot conserved domain. The upstream regulatory element of one Jatropha oleosin gene was also isolated. In silico analysis of this upstream sequence indicated the presence of a number of seed specific regulatory motifs. Transcript analysis for the three oleosin genes revealed tissue-specific differences. Immunoassay by western blot analysis using antibodies against Arabidopsis oleosin and the J. curcas specific oleosin revealed a diversity of oleosins. Proteomic analysis of oil bodies revealed additional minor proteins including caleosin, steroleosin, aquaporin and curcin. Information from these studies is essential for varietal improvement of Jatropha and/or developing oleosin gene fusion technology for expression of novel compounds. In this study 623 nucleotides upstream of the JcOleosin3 gene have been isolated and characterized in transgenic Arabidopsis plant using β-glucuronidase (GUS) as the reporter gene (Chapter 4). Result showed that the JcOleosin3 promoter directs iii expression of the β-glucuronidase gene in seed and pollen but not in leaves, root, stem or flower. In addition, in transgenic Arabidopsis, the JcOleosin3 promoter drives GUS activity at the early seedling stage; this was expected based on earlier reports of expression of Jatropha oleosins. To understand the molecular basis of seed germination, proteomic studies were performed on resting and germinating seeds of J. curcas. Such studies using 2-DE and MALDI-TOF MS revealed the presence of patatin-like lipases in the germinating seeds but not in the resting seeds (Chapter 5). These proteins showed sequence similarity and conserved domains as found in the patatin lipases from Solanum tuberosum and Arabidopsis thaliana. Patatin lipases play a significant role in the mobilization and lipolysis of TAGs during the germination of oil seeds. Further, there is little knowledge on the triacylglycerides (TAGs) lipid cycling in J. curcas., Bioinformatic data-mining confirmed the TAGs cycling in J. curcas to include the expected stages of synthesis, storage and lipolysis of fatty acids. Based on the data-mining results for the presence of various genes for TAG storage, synthesis and utilization, a scheme is presented for TAG cycling in J. curcas. The scheme presented in this study provides essential information required for oil manipulation in J. curcas.Royal Thai Governmen