Agrobacterium tumefaciens - mediated transfer of plant DNA for gene expression analysis.

摘要

Agrobacterium tumefaciens causes crown gall tumor primarily in dicotyledonous plants. The infection mechanism reveals that a portion of bacterial DNA (T-DNA) is transferred into the plant nuclear genome. This transformation system is widely utilized in plant genetic engineering and plant molecular biology.;In this dissertation, two experiments were carried out. First, the copy number, physical structure and F1 segregations of T-DNA copies were analyzed, attempting to understand the mode of T-DNA integration into the plant genome. While the mechanism of Agrobacterium response to plant signals and generation and transfer of T-DNA intermediates through actions of virulence genes is generally understood, the steps leading to the integration of T-DNA into the plant genome are not understood. Toward that understanding, it was essential to investigate the physical and genetic characteristics of the transferred T-DNA within a plant genome. This study, analyzing 75 transgenic tobacco plants, indicated that T-DNAs were present from one copy to a few copies per genome and one to several chromosomal loci were integrated with T-DNA.;Second, vacuole targeting signal of a seed storage protein phaseolin from Phaseolus vulgaris was investigated. Seed storage proteins, comprising approximately 50% of the total protein of the seed, are known to be synthesized in the cytoplasm and transported into vacuole protein bodies. However, the protein signal necessary for the vacuole targeting is not known. Recent studies with yeast have suggested that the targeting signal may be within the polypeptide domain of the seed storage protein. In this study, a probable targeting signal of phaseolin was constructed and then tested in transgenic tobacco plants. A reporter protein, GUS, was translationally fused to seven different lengths of DNA encoding phaseolin protein sequence. These constructions were cloned into a pTJS plant transformation vector. The vacuole targeting of the GUS protein will be investigated to identify the putative vacuole targeting signal of phaseolin.