Date of Award
Doctor of Philosophy
Since the realization of plant genetic modification, transgenic plants have been utilized as platform for the production of valuable recombinant proteins or also known as plant molecular farming (PMF). The plant offers an economical system to reduce cost, the possibility of large scale production and is free of pathogens. The plant also provides versatility o products to accumulate from industrial enzymes, peptides, and pharmaceuticals. Recent advancement of RNA as regulator of gene expression and antiviral responses has shown a new area of research where transgenic plants are used to accumulate therapeutic RNAs. In this study, the reliability of transgenic plants as a platform for the production of industrial enzyme, subunit vaccine and therapeutic RNAs are being evaluated. Key factors that determine the success of molecular farming include 1) robustness of plant genetic transformation, 2) accumulation of large levels of recombinant product, 3) versatility of products to be accumulated, 4) stability of products, and 5) bioactivity of plant-derived products.
The first study demonstrated the accumulation of starch-degrading enzyme, amylopullulanase (APU) derived from bacteria Thermoanaerobacter thermohydrosulfuricus in maize seed. Accumulation of thermostable APU in transgenic maize seed resulted in direct starch processing into glucose and simple sugars without the addition of exogenous enzyme. Subsequent fermentation reaction showed 40.2% conversion of starch into bioethanol. This study showed simplification of starch-based bioethanol production from maize grains using a biotechnology approach.
The feasibility of the plant as the production system for the accumulation of antigen is demonstrated in the second study. Conserved nucleoprotein (NP) gene was expressed in maize seed. This conserved protein showed potential application as a universal vaccine for influenza virus. Our study suggested that maize-derived NP was immunogenic and could induce antibody responses upon administration in pigs and mice. Maize NP seeds remained stable and active at room temperature. This result showed that the plant is able to produce product and maintain immunogenicity.
Thirdly, the plant was utilized as a production system for the accumulation of dsRNA as a vaccine for shrimp viral pathogen. Our result showed the accumulation of long dsRNAs specific to white spot syndrome virus (WSSV) and infectious myonecrosis virus (IMNV). Shrimp bioassay of soybean-derived dsRNA specific to WSSV showed moderate protection against viral challenge and indicated activity of plant-derived dsRNA. While the accumulation levels of dsRNAs in transgenic plants was far from economically efficient, our result showed that the plant was able to accumulate foreign dsRNA. Our result showed that accumulation level needs to be improved for plant use as an efficient platform for accumulation of therapeutics RNA.
Concern over antibiotic or herbicide resistance selectable marker has brought attention for an alternative selectable marker. In the last study, a non-antibiotic selectable marker was assessed as a selection system for maize Agrobacterium-mediated transformation. The ptxD gene conferring phosphite oxidoreducatase enzyme enables transformed cells to utilize phosphite as phosphorous source while non-transformed cells cannot. Phosphite selection resulted in positive identification of transgenic events.
The present study shows the feasibility and reliability of plants as a production system for a variety of products from enzyme, antigen and dsRNA.
Hartinio N. Nahampun
Nahampun, Hartinio N., "Plant molecular farming for the production of industrial enzyme and vaccines" (2015). Graduate Theses and Dissertations. 14962.