Dissertation
CELIAC-SAFE WHEAT, REACHING ONE OBJECTIVE BY TWO APPROACHES, GLUTEN ELIMINATION BY RANDOM AND SITE-DIRECTED MUTAGENESIS, AND DETOXIFICATION BY ECTOPIC EXPRESSION OF "GLUTENASES"
Doctor of Philosophy (PhD), Washington State University
01/2014
Handle:
https://hdl.handle.net/2376/117588
Abstract
Wheat is a major staple food as well as a major cause of gluten syndrome. Gluten-intake in the sensitive individuals elicits various symptoms from gastrointestinal or neurological to fatal. Celiac is one of the most devastating dietary-disorders affects ~1% of the global population. The only effective therapy so far is a strict abstinence diet, which is difficult to follow due to the wide spread application of gluten, misbranding and social constraints. Therefore, in the present study, two approaches were undertaken to develop natural dietary therapies for celiac disease. The first approach is epigenetic elimination of the immunogenic prolamins. Wheat glutens are comprised of high molecular weight glutenins (HMWgs), gliadins and low molecular weight glutenins (LMWgs). The former group largely contributes to the end-use quality and the latter two are the major sources of immunogenic-peptides. Transcription of genes encoding gliadins and LMWgs depends on active demethylation of their promoters in endosperm by a 5-methylcytosine DNA glycosylase/lyase (DEMETER), whereas HMWgs are independent of this regulation. Taking advantage of this differential regulation an effort has been made to characterize mutations in the wheat DEMETER homoeologues using the two reverse genetic procedures, Targeting Induced Local Lesions In Genomes (TILLING) and Transcription Activator-Like Effector Nuclease (TALEN). The second approach involves detoxification of glutens by ectopic expression of "glutenases". A combination of a glutamine-specific barley endoprotease B2 (EpB2) and a post-proline cleaving Pyrococcus furiosus prolyl endopeptidase (PEP) were selected and expressed in wheat endosperm. Wheat transformants showing sizable amount of reduction in Pro/Gln-rich peptides under simulated gastrointestinal conditions are reported. In view of increasing thermostability of EpB2 a structure-based site directed mutagenesis approach was adapted and increase in thermostability by 4°C was reported. The observed increase in the thermostability is not substantial, but this pilot study demonstrated a possibility of obtaining thermostable variant of EpB2.
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Details
- Title
- CELIAC-SAFE WHEAT, REACHING ONE OBJECTIVE BY TWO APPROACHES, GLUTEN ELIMINATION BY RANDOM AND SITE-DIRECTED MUTAGENESIS, AND DETOXIFICATION BY ECTOPIC EXPRESSION OF "GLUTENASES"
- Creators
- NUAN WEN
- Contributors
- Diter von Wettstein (Advisor)Hanu Pappu (Committee Member)Craig Morris (Committee Member)Sachin Rustgi (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 168
- Identifiers
- 99900581444801842
- Language
- English
- Resource Type
- Dissertation