Molecular analysis of genes of white clover (Trifolium repens L.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in biotechnology at Massey University, Palmerston North, New Zealand
The expression of the genes for the small subunit (SSU) of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco), alcohol dehydrogenase (Adh) and lectin in the white clover plant was investigated by Northern analysis, using heterologous plant probes. SSU was shown to be expressed in the leaves/stems, Adh in the roots and lectin in both the leaves/stems and the roots of the mature plant. A series of independent, white clover, leaf/stem and root cDNA libraries was constructed in the lambda vector λgt10 from polyadenylated messenger RNA isolated from mature plants. A number of SSU and Adh cDNA clones was isolated from these libraries and the inserts from these clones were characterized by restriction enzyme mapping and DNA sequence analysis. These clones included a partial SSU cDNA clone from a leaf/stem library and a full length Adh clone from a root library. Two uncharacterized lectin cDNA clones were also isolated from each of the leaf/stem and root cDNA libraries. A fully-representative genomic library was constructed in the lambda vector λEMBL3 from total white clover DNA. This library was screened with the previously isolated white clover SSU and Adh cDNA clones. One SSU and three Adh genomic clones were isolated and the inserts from these clones were characterized by restriction enzyme mapping and Southern blotting. Restriction fragments, to which the corresponding cDNA probe hybridized, were subcloned and characterized by additional restriction enzyme mapping and DNA sequence analysis. The one SSU genomic clone represented a functional white clover rbcS gene, corresponding to the white clover SSU cDNA clone, and was complete with 5' and 3' non-transcribed regions. Conserved sequences were identified in this gene that have been implicated in the regulation of plant gene expression in general and the regulation of rbcS genes in particular. The three Adh genomic clones represented different, non-functional, white clover Adh pseudogenes, each with regions of strong homology to only limited regions of the white clover Adh cDNA clone.