Degree Type

Dissertation

Date of Award

2009

Degree Name

Doctor of Philosophy

Department

Agronomy

First Advisor

Kan Wang

Abstract

The present work addresses fundamental and practical considerations of using maize for production of recombinant proteins. Maize expressing the B subunit of the heat labile enterotoxin (LT-B) of Escherichia coli was used as a model crop. To address the fundamental aspects of the system, the role of LT-B and its native signal peptide (BSP) in plant cells was investigated using translational fusions with the green fluorescent protein. Confocal microscopy showed that fusions carrying BSP or a maize 27 kD γ-zein signal peptide (ZSP) accumulated in the secretory system of Arabidopsis and maize. Fusions lacking a signal peptide accumulated in the cytosol or nucleus. Understanding how plants process bacterial signal sequences can aid in developing effective strategies for producing recombinant proteins in plants.

To address the practical aspects of the system, fractionation of transgenic LT-B maize by wet milling was performed using traditional (SO2+lactic acid) or water steeping. Functional LT-B was found at highest concentration in the fine fiber, lowest in germ and starch, and with highest total LT-B recovery from water steeping. Wet milling of LT-B maize with water steeping is suitable for enrichment of a high-value protein in fine fiber; co-utilization of germ and starch for traditional uses is feasible.

A continuous in vitro endosperm callus culture (ENC) of Hi II maize was developed for functional analysis of seed-specific expressing genes. Morphological and biochemical characterization of ENC showed presence of aleurone and starchy endosperm cell types that resemble those of developing endosperms. ENC cells synthesize starch, zein storage proteins, and possess active starch metabolism enzymes. While the current ENC needs improvement for effective transient analysis of promoter/gene expression, it is useful for endosperm-targeted transgene analysis in stably-transformed lines of genotype Hi II.

We expressed an endo-1,4-β-glucanase from Ruminococcus albus in maize for potential use in bioethanol production. Molecular characterization showed successful transgene integration in recovered lines. In-depth biochemical analysis indicated that current standard assay systems need improvement for thoroughly characterizing recombinant endoglucanase.

This work illustrates that maize, an important food, feed and feedstock crop, can be used for efficient production of recombinant proteins in seeds.

Copyright Owner

Lorena Beatriz Moeller

Language

en

Date Available

2012-04-28

File Format

application/pdf

File Size

196 pages

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