Date of Award

Spring 1991

Project Type

Dissertation

Program or Major

Botany

Degree Name

Doctor of Philosophy

First Advisor

Subhash C Minocha

Abstract

Ornithine decarboxylase (ODC), arginine decarboxylase (ADC), and S-adenosyl-methionine decarboxylase (SAMDC) are the three key regulatory enzymes for polyamine (putrescine, spermidine, and spermine) biosynthesis. In order to gain more insight into the relationship between polyamine metabolism and other physiological processes, research was undertaken to obtain increased putrescine biosynthesis in tobacco by overexpression of a murine ODC cDNA.

Both a full-length and a truncated murine ODC cDNA were cloned into a binary expression vector containing the Cauliflower Mosaic Virus (CaMV) 35S promoter. Using standard leaf-disc transformation procedures, transgenic tobacco plants containing either the full length or the truncated ODC cDNA were obtained. Presence of the murine ODC cDNA as well as transcription were confirmed via Southern and Northern blotting. Western blot analysis identified a polypeptide unique to the transformed plants which immunoreacted with anti-ODC antibody.

A series of enzyme assays were done to differentiate between native and murine ODC activity. Assays were run at the pH optima for native ODC (pH 8.2) and murine ODC (pH 6.8). At pH 6.8, there was very little activity in the control plants, but a significantly higher activity in the transformed plants. Difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC activity, completely inhibited ODC activity in the transformed plants at pH 6.8. However in the control plants at pH 6.8 and both the control and transformed plants at pH 8.2, DFMO only inhibited ODC activity by approximately 30-50%. Almost 100% inhibition of ODC activity by immunoprecipitation of ODC protein with anti-ODC antibody was observed in the transformed plants, at pH 6.8. The results show clearly that the activity of murine ODC can be detected and quantified even in the presence of the plant ODC. The transgenic plants containing the truncated ODC cDNA always had several fold higher activity than those containing the full-length cDNA.

Transgenic plants containing the truncated ODC cDNA contained 10-12 times the levels of putrescine than the control plants. Transgenic plants containing the full-length cDNA contained 4-5 times the level of putrescine as compared to the control. In addition to increased levels of putrescine, there was an amine-containing compound unique to the transformed plants with a retention time very similar to putrescine.

Download

Share

COinS