Date of Award
12-17-2012
Document Type
Dissertation
Degree Name
Molecular Biosciences, Ph.D.
First Advisor
Elizabeth Hood
Committee Members
Brett Savary; Carole Cramer; Gregory Phillips; John Howard
Call Number
LD 251 .A566d 2012 Y32
Abstract
One of the greatest barriers to produce biofuels from lignocellulosic material is efficient cellulose depolymerization by cellulase activity. Expansin is a protein that is active in cell wall expansion in growing regions of most plants. Because of this unique activity, expansin synergistic activity with cellulase is believed to lower the depolymerization barrier. Over-expression of expansin in the maize production system is designed to produce sufficient protein 1) to study its physiological effects, and 2) to assess its ability to serve as an industrial enzyme for applications, particularly in biomass conversion. Transgenic lines of maize that express the cucumber expansin gene have been recovered. The gene is expressed from an embryo-preferred promoter, globulin-1, and the protein is targeted to three sub-cellular locations: the cell wall, the vacuole and the endoplasmic reticulum (ER). About four hundred plants were recovered and first generation seed was harvested from them. Levels of expansin in these seeds have not been assessed because of the absence of an efficient expansin activity assay system. The traditional activity assay for expansin is cumbersome and slow, thus it is not practical for screening large numbers of samples. Therefore, a novel high-throughput expansin assay which measures the amount of glucose from microcrystalline cellulose by expansin synergistic activity with cellulase was developed. To develop the expansin assay, a glucose assay kit from Sigma Chemical Co. was modified and optimized to detect expansin activity (release of glucose from cellulose), using enriched cucumber expansin in this novel assay. The novel expansin assay was used to identify high-expressing transgenic maize lines. First generation seeds from out-crossed T0 plants of cell wall, ER and vacuole targeted lines were screened and ER lines showed the best expression. Among the ER lines, two specific transgenic plant lines, BCG01 and BCG04 showed highest expression. To choose the best plants in the events BCG01 and BCG04, single seed assays were conducted and plant 10 of event 1 and plant 12 of event 4 were chosen as the best expressers for further development. Enriched protein extract of ER targeted transgenic maize lines was used to perform digestion tests in combination with cellulases on several cellulosic samples. In this study, a novel high-throughput expansin assay was developed and transgenic maize lines were screened using this assay. Activity on pretreated lignocellulosic material of recombinant corn expansin from transgenic maize was confirmed. Activity of recombinant corn expansin from ER-targeted lines can now be used for breeding to increase expansin expression for use in the biomass conversion industry.
Rights Management
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Yoon, Sangwoong, "Expansin Synergy with Cellulases: Development of a Transgenic Maize System for Expansin Production and Evaluation of Synergistic Activity on Lignocellulosic Substrates" (2012). Student Theses and Dissertations. 829.
https://arch.astate.edu/all-etd/829
