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Type: Theses
Title: Investigating the synthesis and regulation of (1,3;1,4)-β-glucan biosynthesis
Author: Dimitroff, George
Issue Date: 2016
School/Discipline: School of Agriculture, Food and Wine
Abstract: Cereals such as rice (Oryza sativa (Os)), barley (Hordeum vulgare (Hv)) and sorghum (Sorghum bicolor (Sb)) provide a considerable portion of our daily energy requirements. Their cell wall constituents, such as (1,3;1,4)-β-glucan, survive relatively intact through much of the upper human digestive system to reach the colon, where they are fermented by a range of commensal microorganisms. The products of this fermentation help reduce blood cholesterol levels and ameliorate diseases including coronary heart disease, type II diabetes and colorectal cancer. Efforts have therefore been directed toward understanding the regulation and mechanism of (1,3;1,4)-β-glucan biosynthesis to enhance the human health potential and industrial utility of cereal grain. Numerous reports suggest that the CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene encodes the synthase responsible for producing the majority of (1,3;1,4)-β-glucan in cereals. These synthase genes contain species-specific polymorphisms that have been shown to influence the amount and structure of (1,3;1,4)-β-glucan produced when they are expressed heterologously in Nicotiana benthamiana and barley grain. Here, a chimeric approach exchanged sections of the barley (Hv) and sorghum (Sb) CSLF6 synthases to identify regions influencing (1,3;1,4)-β-glucan production and structure. Using this approach an 80 amino acid stretch, which contains the conserved TED and QxxRW motifs, was shown to be responsible for much of the difference in (1,3;1,4)-β-glucan production and structure between the barley and sorghum synthases. Of the six amino acid polymorphisms contained within this section, one affected polysaccharide structure whilst another dictated the amount of (1,3;1,4)-β-glucan. Co-expression in N. benthamiana was used to investigate CSLF6 modulation and complex formation. Results from a variety of chimeric, truncated and mutated constructs suggest that a highly variable section of unknown function, termed the class-specific region (CSR), and the NH2-terminal region of CSLF6 are separately able to mediate complex formation and increase (1,3;1,4)-β-glucan production. Expression of a construct missing the CSR indicated that the region was not structurally or functionally required for (1,3;1,4)-β-glucan synthesis in N. benthamiana. A PilZ domain responsible for cofactor binding and cellulose synthase activation in bacteria was also identified at the COOH-terminal end of the NH2-terminal region of CSLF6, and was shown to influence (1,3;1,4)-β-glucan production. Overall, the results presented here have furthered our understanding of the action of the CSLF6 isoform of the (1,3;1,4)-β-glucan synthase enzyme. This brings us closer to having the capacity to precisely control the synthase’s function, and allowing the prospect of manipulating cereal tissues to contain the optimal amount of (1,3;1,4)-β-glucan with a defined structure for specific human health and industrial applications.
Advisor: Burton, Rachel Anita
Little, Alan
Fincher, Geoffrey Bruce
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2016.
Keywords: (1,3;1,4)-β-glucan
chimera
cereal
dietary fibre
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals
DOI: 10.4225/55/5af3f236b6546
Appears in Collections:Research Theses

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