Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

Jesse M. Jaynes


The winter flounder, Pseudopleuronectes americanus, can survive in sea water at temperatures below freezing by producing antifreeze-proteins (AFPs) which depress the freezing point of their cellular fluids. The antifreeze gene (IIA7 cDNA) encodes a 91 amino acid preproprotein which is processed to a mature protein of 53 amino acids. This gene encoding only the mature antifreeze-protein, including a start methionine, was cloned into a plasmid which allowed enhanced expression from a double CaMV 35S promoter. The AFP gene construct was subcloned into both the intermediate vector pMON200 and the binary vector pBI121. After triparental mating and infection of tobacco leaf discs with Agrobacterium tumefaciens containing either pBI121AF or pMON200AF, transgenic plantlets were obtained which were kanamycin resistant and GUS positive. Southern analysis confirmed the presence of single-copy gene integration. The transcription levels of the antifreeze gene were significantly higher than those of a single CaMV 35S promoter and a Western blot confirmed the synthesis of the immunoreactive 5.5 kd antifreeze-protein in the transgenic tobacco tissues. Several individual plant seedlings which were kanamycin resistant were selected and tested for frost tolerance. At least 30% more transgenic plants survived than the control plants. These results confirmed the ability of this fish protein to confer increased frost-tolerance to plants.