The isolation and characterization of a senescence associated nodulin cDNA.

Abstract

The goal of this thesis is to study cellular senescence with the use of the determinate nodule of soybean as the model system. The determinate nodule is an appropriate model system for the study of senescence due to the homogeneity of infected cells of various developmental stages within the organ. In this model system consisting of Glycine max (L.) Merrill, Maple Arrow, infected with Bradyrhizobium japonicum strain 61A76, we showed that nodule senescence occurs late in nodulation, at the time of flowering and pod filling. We also demonstrated that nodule senescence can be induced with the addition of 20mM ammonium nitrate. The strategy to study the molecular mechanisms of nodule senescence involves the isolation of senescence-associated nodulin (SAN) cDNAs by the differential screening of a cDNA library representing mRNA from senescent nodules. This library constructed in the $\lambda$gt11 vector is screened with two bulk probes made from the PCR amplification of the cDNA inserts of libraries in the $\lambda$gt10 vector: one representing mRNA of healthy nodules that are actively fixing nitrogen and the other of senescent nodules. In this thesis, one SAN cDNA, clone 8-4 was characterized through a Northern analysis to demonstrate the up-regulation of the corresponding gene with senescence. In the natural senescent system, SAN8-4 expression was demonstrated to increase with nodule senescence. A search through nucleotide and protein sequence banks demonstrates that SAN 8-4 may be a novel F3H related protein. (Abstract shortened by UMI.)