Belfield, Graham Paul (1986) The genetic manipulation of a cDNA encoding the seed storage protein legumin a to alter its amino acid composition. Masters thesis, Durham University.
The seeds of lugumes are eaten throughout the world, constituting a major input of dietary protein. There can be problems of malnutrition in the more deprived areas where they provide the main source of protein, for though legume seeds can be very rich in protein, they are deficient in some amino acids, mainly methionine, that are essential for a healthy diet. Recombinant DMA technology may offer a solution to this problem by introducing DNA encoding these deficient amino acids into seed storage protein genes, which on reintroduction into the host plant- could be grown as a more nutritional crop. The 2s storage proteins of the Brazil nut contain a very high proportion of methionine, therefore DNA encoding 2s protein could be introduced into the sequence of legume storage protein genes. It was proposed that this be attempted, and any constructions that should be produced could be cloned into yeast to detect expression of the mutated genes. Attempts were made to construct and isolate pUCl8 vector clones of Brazil nut DNA, to determine and attempt a rationale for the insertion of DNA from these clones into sequences encoding legumin A by site directed mutagenesis, and to detect the formation of Brazil nut - legumin DNA constructs with radiolabelled probes DNA probes and agarose gel electrophoresis. Initial steps were taken to perform a similar mutation of a vicilin cDNA. Two pUCl8 clones of Brazil nut DNA; - pBnA and pBnB were created and isolated. Clones of legumin - Brazil nut DNA;- pGPBl were constructed based on an insertional mutation of the legumin cDNA construct pJYB with Brazil nut DNA from the clone pBnA. They were isolated from the in – situ hybridisation of transformed cells with radiolabelled DNA. Clones of pGPBl were found by gel electrophoresis and Southern hybridisation to contain the BnA insertion in the correct orientation. Time constraints prevented the cloning of pGPBl into yeast. The choice of legumin mutation sites was discussed and the rationale adopted justified on the grounds of restriction site analysis and the restraints imposed by legumin solubility and protein structure. The Results and problems encountered were discussed in some detail. It was suggested that further work should attempt the expression of pGPBl in yeast.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||08 Feb 2013 13:48|