Construction of a Synthetic Dihydrofolate reductase gene

Includes bibliographical references (pages [63]-66)The goal of this research was to improve Dihydrofolate reductase (DHFR) as a selectable marker in a maize transformation system. To do this, molecular, tissue culture and transformation techniques were used to transiently express several DHFR proteins and mRNAs. Three DHFR genes were used in this study to code for mutant enzymes to which the selective agent methotrexate, a folate analog, has low affinity. The bacterial DHFR used in this study has virtually no affinity to methotrexate and has been shown to function in dicots when expressed in a viral vector. The synthetic gene is based upon the sequence the bacterial gene but utilizes the preferred codon usage of maize which favors s C or G nucleotide in the "wobble" position. An altered mouse gene was used as a positive control since it has already been demonstrated to express in stable plant transformants and inherited by their progeny. The mouse, bacterial and synthetic dihydrofolate reductase genes were cloned into expression cassettes using the Cauliflower Mosaic Virus 35S promoter and the Nopaline synthase polyadenylation signal. These DHFR expression vectors were electroporated into maize and tobacco protoplasts which were assayed for DHFR enzyme and mRNA. The DHFR enzymes was also assayed in DH5-α E. coli cells containing the constructs. The DHFR enzyme could not be confirmed by activity staining in either extracts from the protoplasts or the DH5-α cells. The mRNA from protoplasts did not show the presence of DHFR mRNA when Northern blot hybridizations were performed.M.S. (Master of Science