Mühling, Martin (1996) Molecular studies on seed proteins from mature seeds of Phaseolus Vulgaris L. Masters thesis, Durham University.
In order to elucidate the question of the presence or absence of legumin in the seeds of Phaseolus vulgaris L. it was decided to investigate the globulin fraction of seed proteins from mature seeds of French bean. A legumin protein was partially purified and the N- terminal sequences of both subunit polypeptides of a 75 kDa legumin subunits determined, which proved unequivocally that these polypeptides are subunits of a P. vulgaris legumin. The N-terminal amino acid determination of further five disulphide-linked polypeptides of lower Mr revealed that all these polypeptides may well derive from the 75 kDa P. vulgaris legumin as the N-terminal amino acids showed complete sequence homology. The size differences of these legumin subunits might be due to peptide cleavage at the C-terminal end of the legumin a-chains. Potential mechanisms for this catalytic event are discussed. In addition, a second protein. Glycoprotein I, an albumin type seed protein, was isolated from the albumin fraction of the seed proteins of P. vulgaris and the N-terminal amino acids of its subunits were determined. The obtained sequence data of the three different subunits revealed high similarity to the hyper-variable region of legumin J and K from pea. Whilst it is not inconceivable that both polypeptides represent independent protein subunits reflecting the same evolutionary mechanisms to increase the nitrogen content of storage proteins, based on the sizes of the Glycoprotein I subunits and the sequence similarity to the hyper-variable region of a-chains of legumins the hypotheses is discussed that Glycoprotein I may derive from a P. vulgaris legumin by cleavage of peptides from the C-terminal end of the a-chain and formation of a new polypeptide, Glycoprotein I, with new solubility characteristics (water-soluble like albumins).Furthermore, Glycoprotein I was investigated during seed germination by immunodetection with antibodies raised against the purified protein. The results showed that Glycoprotein I serves as a reserve protein in an early stage of seed germination. In order to obtain extensive sequence information on legumins from P. vulgaris it was decided to attempt to amplify a legumin gene fragment from genomic DNA of P. vulgaris using the polymerase chain reaction technique. Southern analysis of PGR products using a pea-legumin as a probe gave strong evidence for the successful amplification of legumin encoding genes from genomic P. vulgaris DNA. However, neither the cloning of these PGR products nor the repeats of this PGR experiment were successful. A new PGR approach to amplify legumin gene fragments from genomic DNA of P. vulgaris using an oligonucleotide sequence for the reverse primer designed on the basis of the N-terminal amino acid sequence of the β-chain is discussed. The nucleotide sequence of a PGR artefact of one of the unsuccessful repeats of the PGR experiment showed sequence similarity to Arcelin 2, a gene found in wild types of P. vulgaris. Arcelin proteins provide the plant with resistance against an important pest weevil, but have never been observed in cultivars of P. vulgaris. Arcelin genes are thought to have been either totally lost or inactivated during domestication of the French bean. Further isolated and identified proteins from the albumin fraction of seed proteins from P. vulgaris include a superoxide dismutase (SOD). The determination of the N-terminal amino acids of the subunit poypeptide provides the first reported sequence information of a SOD in mature seeds of higher plants. The potential localisation within the seed tissue and the putative role of the presence of a SOD in mature seeds during dormancy is discussed. Two more polypeptides were isolated from the albumin fraction and identified by N- terminal sequencing as chitinases. Whereas pvchi25 showed high sequence similarity to class n chitinases, pvchi32 was shown to be a class III chitinase. Pvchi32 provides the first reported class HI chitinase in mature seeds of higher plants and the second so far described glycosylated chitinase. The potential use of these seed chitinases in crop protection is discussed. Furthermore, the enzymatic activity of both enzymes was tested as well as cross-reactivity to antibodies raised against ethylene-induced bean leaf chitinases. Pvchi25 was observed during seed germination by immunodetection confirming its role in the protection of the seed during dormancy and early germination.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||24 Oct 2012 15:10|