Transgenic analysis of ORF239: Cellular localization in tobacco plants, protoplasts and yeast

Delkin Orlando Gonzalez, Purdue University

Abstract

In Phaseolus vulgaris L, cytoplasmic male sterility (CMS) has been associated with a dominant mitochondrial mutation known as pvs-orf 239. Histological evaluation of developing microspores using fluorescense microscopy shows that callose deposition is abnormal, and immunocytochemistry studies utilizing light microscopy reveal that ORF239 is localized within the callose layer and developing cell walls of the sterile bean line (Abad et al., 1995). These data suggest transport of the ORF239 protein product from the mitochondrion to the periphery of the cell. In an attempt to find a biological system that allows study of mitochondrial transport, I evaluated three systems: tobacco transgenic plants, tobacco protoplasts and the yeast Pichia pastoris. Tobacco plants transformed using Agrobacterium-mediated transformation techniques provided little information about this phenomenon due to lack of a strong pollen-specific promoter. Transformation of tobacco protoplasts by electroporation provided a better system, and allowed confirmation of mitochondrial localization of transgene products. However, technical limitations were found using the β-glucuronidase reporter gene to follow ORF239 transport. Analysis of transformed Pichia pastoris cells with the pvs-orf239 sequence and truncated amino and carboxy versions, provided a more powerful system to study mitochondrial protein transport. Both the entire ORF239 and its carboxy terminus were localized at the periphery of the cell and cell wall suggesting that mitochondrial transport had occurred. Since the carboxy end was apparently sufficient for transport, future studies will be focused on designing additional truncations and on refined mutational analysis in this region using Pichia as a model system.

Degree

Ph.D.

Advisors

Mackenzie, Purdue University.

Subject Area

Genetics|Molecular biology

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