Title page for ETD etd-07092012-131903


Type of Document Master's Thesis
Author Dere, Madhavi Suresh
Author's Email Address msdere@vt.edu
URN etd-07092012-131903
Title GENETIC TRANSFORMATION OF SWITCHGRASS (Panicum virgatum L.) WITH ENDOGLUCANASE GENE AND CHARACTERIZATION OF PLANTS WITH ENDOGLUCANASE TRANSGENE
Degree Master of Science
Department Horticulture
Advisory Committee
Advisor Name Title
Zhao, Bingyu Committee Chair
Maroof, M. A. Saghai Committee Member
Veilleux, Richard E. Committee Member
Vinatzer, Boris A. Committee Member
Keywords
  • genetic transformation
  • hygromycin
  • GUS assay
  • Panicum virgatum
  • Switchgrass
  • chloroplast
  • RuBisCO
  • signal peptide
  • Hypocrea jecorina
  • endoglucanase
  • TAIL PCR
  • pSQ5
  • activation tagging
  • GFP
Date of Defense 2012-06-22
Availability unrestricted
Abstract
As a warm season grass native to the North American continent, switchgrass is considered as one of the most promising biofuel crops in the USA. It is a C4 plant that makes it energy efficient. Switchgrass has a deep root system that allows it to grow on marginal land with low water and nutrient input. Switchgrass has been used as a forage crop and its use for biofuel will not affect food security. Biofuels are more environment-friendly than fossil fuels as they do not produce net greenhouse gases. However, the problem of high cost of production per unit for biofuel has to be overcome if we want to replace fossil fuels with biofuels. One of the major factors related to the high cost of biofuel are the expensive cellulase enzymes used in the pretreatment of feedstock. Endoglucanase is the key enzyme used for breaking down cellulose before fermentation. Currently, endoglucanase is produced from engineered E. coli or yeast strains, which is still expensive for enzyme production and purification of industrial scales. Expression of endoglucanase in plants has been previously reported. However, there are no reports of transgenic switchgrass producing cellulase enzyme. In this study, the catalytic domain of beta-endoglucanase gene was codon-optimized and synthesized based on the cDNA cloned from Hypocrea jecorina. Rice RuBisCO small subunit targeting signal peptide was fused to the N-terminus of the beta-endoglucanase gene, which was expected to target the fusion protein to chloroplast. This subcellular compartment targeting could minimize negative effects on cell function and plant development. The endoglucanase gene was cloned with maize ubiquitin promoter in a modified binary vector pCambia 1305-2 and transformed into switchgrass genotype HR8 by using Agrobacterium tumefaciens. In this study, I generated five independent transgenic switchgrass lines and they were confirmed by growing on the selection agent hygromycin, GUS assay, PCR amplification, southern blotting hybridization, for the presence of hygromycin and endoglucanase genes. However, based on RT-PCR analysis, only two transgenic lines were confirmed to produce mRNAs of the endoglucanase gene. These two transgenic lines were further characterized for their agronomic traits and the chlorophyll contents. Our results suggested that expression of endoglucanase in switchgrass could reduce chlorophyll content and affect plant development. Nevertheless, in this study, we demonstrated that a fungal endoglucanase gene could be expressed in switchgrass transgenic plants, though the gene expression level and the subcellular localization need to be carefully regulated in order to minimize the toxic effect of endoglucanase on plant cells.

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