MOLECULAR CLONING IN MAIZE

Recombinant DNA and plant tissue culture selection techniques will be used to develop modified versions of Cauliflower mosaic virus (CaMV)DNA. It is intended that the final modified version be capable of transferring new DNA sequences or genes into maize tissue culture cells where stabilization and expression of such genes could occur. Our approach is to insert a bacterial drug resistance marker gene into a nonessential region of the CaMV genome using synthetic primers, in vitro strand synthesis, and M13 cloning techniques. The marker gene will be introduced into plant tissue culture cells and drug-resistant cells selected. We will further modify the marker CaMV genome by in vitro mutagenesis and cloning into E. coli using the plasmid pBR322 to identify other potentially nonessential regions of the CaMV genome. These regions will be deleted to the extent possible; their location, size, and the sequence of the new constructions will be determined by M13 sequencing. The drug resistance marker will be maintained by selection in E. coli and confirmed by selection in plant cell cultures. We will then insert new DNA sequences into appropriate deleted regions and transform plant cells by selection for drug resistance. The fate and expression of this second gene in plant cells will be studied. Our goal is to introduce a gene for the maize storage protein, zein, into the transformation vector and to reintroduce this gene back into maize tissue culture cells. The ability to select for a linked marker gene will allow us to address many questions about what happens to the zein gene in such gene transfer experiments.