SOME MOLECULAR ASPECTS DURING SEED DEVELOPMENT IN SOME LEGUMES

Abstract

The present study deals with the genes involved in carbon fixation during photosynthesis in higher plants. The practical work undertaken in this study can be divided into three main parts. The first part includes cloning, identification and sequencing of cDNAs for Calvin cycle enzymes, to obtain a complete set of homologous full-size molecular probes for ail of the Calvin cycle enzymes from a eDNA library of spinach (Spinacia oleracea L. cv. Monopa). From the twelve molecular probes, the probes ofphosphoribulokinase (PRK), fructose-1,6-bisphosphatase (FBPase) and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco SSU) (RbcS) were isolated and characterized as part of this thesis. The clones ofFBPase and RbcS were prepared for the first time. In the second part, seeds of pea (Pisum sativum L. cv. Rosa Krona) and bean (Phaseolus vulgaris L. cv Nikarkoenigin) were seeded in field soil. The growth of the two plants was followed until the end of their life cycle. During the seedling and vegetative stages, samples were harvested from roots, cotyledons and leaves. During the embryogenesis stages, samples were taken from leaves, pericarps and developing seeds. Each sample was divided into two parts. From the first part, total proteins were extracted and assayed quantitatively, and the enzymatic specific activity of each of the Calvin cycle enzymes was followed in each organ throughout the growth stages studied. In the third part, total RNA was extracted, denatured and immobilized by dot blotting. The molecular probes of the Calvin cycle enzymes were radioactively labeled, and used to hybridize the immobilized mRNA. This was carried out to study the gene expression of the Calvin cycle enzymes in each organ. Some of the Calvin cycle enzymes, ribulose-1,5-bisphosphate carboxylase (Rubisco), phosphoribulokinase (PRK), NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and NADP-dependent fructose-1,6-bisphosphatase, which are localized exclusively in the chloroplast, showed remarkable levels of specific activity and gene expression in the root which, is a non-photosynthetic organ. In addition, remarkable levels of gene expression and specific activity oftransketolase (TKL), ribulose-5-phosphate 3- epimerase (RPE) and ribose-5-phosphate isomerase (RPI), that are common to both the Calvin cycle and the oxidative pentose phosphate pathway (OPPP), were detected in both photosynthetic and non-photosynthetic organs.