Development of marker-free transgenic method and characterization of Arabidopsis VEC1 and VBP1 associated with vesicular trafficking

Abstract

For the production of transgenic plants with commercial and environmental applications, both development of transgenic methods and exploration of genes with utilizable traits should be performed in parallel. In my research, I aimed at, first, the development of revised co-transformation method using negative selectable marker gene to segregate out the progenies with selectable marker genes easily and, next, exploration of genes involved in stress response in Arabidopsis. A negative selectable marker gene, codA, was successfully co-transformed with GUS reporter gene to develop selectable marker gene-free transgenic plants. The pNC binary vector contained a T-DNA harboring the codA gene next to the nptii gene, while a second binary vector, pHG, contained a GUS reporter gene. Tobacco plants (Nicotiana tabacum Samsun NN) were co-transformed via the mixture method with Agrobacterium tumefaciens LBA4404 strains harboring pNC and pHG, respectively. Seeds harvested from the co-transformants were sown on germination media containing 5-fluorocytosine (5-FC). Analysis of the progeny by GUS staining and PCR amplification revealed that all of the 5-FC-resistant R1 plants were codA-free, and that the codA gene segregated independently of the GUS gene. Because codA-free seedlings developed normally on 5-FC-containing medium, co-transformation with negatively selectable markers is a viable method for the production of easily distinguished selectable marker gene-free transgenic plants. While searching for the genes applicable for the production of plants with new traits such as stress-tolerance, I identified and characterized the Arabidopsis VEC1 (Vesicular trafficking-associated C2 domain protein), a novel plant-specific protein that is unusually small in size and has a unique primary structure. VEC1 is highly abundant in glycine, tyrosine, and proline. Their compositions correspond to about 50% of total amino acids composition and also almost all of them are positioned i...