Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects

• Published in: Transgenic research 2000-02, Vol.9 (1), p.43-54
• Main Authors: Dai, Z, Hooker, B.S, Anderson, D.B, Thomas, S.R
• Format: Article
• Language: English
• Subjects: Acidothermus cellulolyticus; Actinomycetales - genetics; AE1 protein; Blotting, Western; Cellulase - genetics; Cellulase - metabolism; cellulases; Chloroplasts; Chloroplasts - genetics; Chloroplasts - metabolism; Chloroplasts - ultrastructure; E1 protein; endoglucanase E1; Gene Expression Regulation, Plant; gene transfer; genetic transformation; Hydrogen-Ion Concentration; Immunohistochemistry; Microscopy, Electron; Nicotiana - enzymology; Nicotiana - genetics; Nicotiana tabacum; Photosynthesis; Plant Extracts - metabolism; Plant Leaves - genetics; Plant Leaves - metabolism; Plant Leaves - ultrastructure; Plants, Genetically Modified; Plants, Toxic; Promoter Regions, Genetic; Ribulose-Bisphosphate Carboxylase - genetics; Transgenic plants
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1023/A:1008922404834

The expression of the Acidothermus cellulolyticus endoglucanase E1 gene in transgenic tobacco (Nicotiana tabacum) was examined in this study, where E1 coding sequence was transcribed under the control of a leaf specific Rubisco small subunit promoter (tomato RbcS-3C). Targeting the E1 protein to the chloroplast was established using a chloroplast transit peptide of Rubisco small subunit protein (tomato RbcS-2A) and confirmed by immunocytochemistry. The E1 produced in transgenic tobacco plants was found to be biologically active, and to accumulate in leaves at levels of up to 1.35% of total soluble protein. Optimum temperature and pH for E1 enzyme activity in leaf extracts were 81 degrees C and 5.25, respectively. E1 activity remained constant on a gram fresh leaf weight basis, but dramatically increased on a total leaf soluble protein basis as leaves aged, or when leaf discs were dehydrated. E1 protein in old leaves, or after 5 h dehydration, was partially degraded although E1 activity remained constant. Transgenic plants exhibited normal growth and developmental characteristics with photosynthetic rates similar to those of untransformed SR1 tobacco plants. Results from these biochemical and physiological analyses suggest that the chloroplast is a suitable cellular compartment for accumulation of the hydrolytic E1 enzyme.