No description available.

This study has demonstrated and investigated the expression of a cDNA, coding for the pea seed storage protein vicilin, in the yeast, Saccharomyces cerevisiae. The cDNA was contained in the plasmid pLG1.63 and has been characterised and sequenced. The sequence showed that the cDNA coded for a 47KDa type of vicilin with a putative 24 amino-acid signal peptide, a proteolytic cleavage site and one glycosylation signal. The cDNA was cloned into two yeast expression vectors. The first utilised the GALIO promoter rendering expression of the cDNA inducible galactose, the construct was called pDUB2300. The second construct, pDUB2302, placed the cDNA under the control of the PGK promoter, rendering the cDNA constitutively expressed. When transformed into yeast, both constructs produced an immunoreactive vicilin species of M(_r) =49KDa. In the case of pDUB2302 the protein was produced at up to 5.5% of total cell protein. The protein was shown to be associated with a particulate fraction and displayed altered precipitation characteristics when compared with pea vicilin. By using tunicanydn and N-glycosidase, the protein was shown to be unglycosylated. Partial purification and (^35)S-methionine labelling demonstrated that the signal pep tide remained uncleaved. Cell fractionation studies indicated that vicilin was enriched in the yeast microsomal fraction, suggesting that vicilin was located in the EH. This was confirmed electron microscopy of immuno-gold labelled yeast which showed vicilin associated with the ER. The electron micrographs also suggested that a small proportion of the protein might be reaching thecolgi apparatus and the vacuole membrane. The presence of specific cleavage products on some western blots suggested that vicilin possessed a cleavage site for a yeast protease, though whether this was the same site as the pea proteolytic cleavage site was not determined. The pattern and nature of the expression of vicilin from this cDNA was discussed in the context of heterologous protein expression in yeast in general and plant storage protein expression in yeast in particular.

Native Glycoprotein II (Phaseolin, G-II), the major 7S storage protein from <i>Phaseolus vulgaris</i> seeds, var. 'Processor' is known to be resistant to <i>in vitro</i> proteolysis by most endopeptidases. On sequential treatments with pepsin and a mixture of trypsin and chymotrypsin, the sub-unit polypeptides of G-II were split midchain. The fragments produced however, retained reactivity with the antibody raised against native G-II quantitatively. When measured by rocket immunoelectrophoresis, the extent of <i>in vitro</i> degradation of G-II by these endopeptidases was negligible. This procedure was used for monitoring the <i>in vivo</i> or <i>in vitro</i> degradation of G-II by gut enzymes other than trypsin or chymotrypsin. Diets containing 10% of a highly purified G-II preparation, did not support growth of rats adequately. Faecal N outputs were elevated and the true N digestibility based on Kjeldhal estimation was only 37%. In contrast, the true GII-N digestibility, based on immunological estimations, was high. It is suggested that G-II and/or its limited breakdown fragments (by trypsin or chymotrypsin) are stimulants of endogenous N secretion in the small intestine. The higher extent of the degradation of G-II in the small intestine of rats <i>in vivo</i> than that obtained by pure endopeptidases <i>in vitro</i> suggested the presence in this tissue of other enzymes capable to act upon and modify the structure of G-II, prior to the action of trypsin and chymotrypsin. These other modifying proteolytic enzymes render the G-II molecule more negatively charged and more susceptible to the subsequent action of trypsin and chymotrypsin. It is suggested that protease content and the ratio of the concentration of the GII-modifying protease(s) to that of trypsin and chymotrypsin may vary appreciably along the small intestine. Accordingly, the dependence of the degradation of G-II <i>in vivo</i> on the competition between all the enzymes capable of attacking it during its passage through the gut may explain the variability of GII breakdown <i>in vivo</i>.

MSCAGR (Plant Production) / Department of Plant Production / Tepary bean (Phaseolus acutifolius) is an important food legume originating from South America and the South-western parts of the United States. The crop is produced in many countries worldwide including South Africa. It is highly tolerant to drought and the seed contains a wide range of vitamins, minerals and protein of high nutritional quality. The genetic base of tepary bean is narrow but can be widened by chemical mutagenesis. However, there are no reports on the impact of chemical mutagenesis on the root nodulation and seed storage proteins in tepary bean. Therefore, this study was designed to examine root nodulation attributes and seed storage proteins of three tepary bean genotypes in the early mutagenic generations (M2 to M4) derived through treatment with varying doses (0.0, 0.5, 1.0, 1.5 and 2.0 v/v) of ethyl methanesulfonate (EMS). The experiment on root nodulation attributes was laid out as a 3 x 5 x 3 (genotypes x EMS doses x mutant generations) factorial design replicated three times. At harvest, shoot height (SHT), primary root length (PRL), dry weights (shoot, root and nodule), number of nodules per plant (NNP) and grain yield components such as the number of pods per plant (NPP) and number of seeds per pod (NSP) were measured. Highly significant (P≤0.01) dose effects were observed for SHT, PRL, shoot dry weight (SDW) and root dry weight (RDW). Highly significant (P≤0.01) interaction effects of mutant generation x genotype x dose were observed for NSP. A highly significant (P≤0.01) positive linear relationship was observed between the NNP and nodule dry weight (NDW). Increase in the PRL suggested that tepary bean mutants could be important in drought tolerance. EMS treatment led to an enhanced partitioning of dry matter (assimilates) to the shoots and roots. There was a three fold increase in most of the root nodulation traits at the 0.5% EMS dose.The Kjeldahl method was used for crude protein determination whereas the sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS PAGE) was utilized in determining the protein banding patterns of the bean. There were highly significant (P≤0.01) differences among the genotypes in crude protein accumulation. Highly significant (P≤0.01) mutant generation x genotype x dose were observed for seed protein accumulation. ‘Genotype 3’ attained the highest protein content (24.23%) at 1.5% EMS dose in the M4 generation. EMS doses ≥0.5% positively stimulated protein accumulation in all genotypes but high EMS doses (2.0%) depressed protein content. There were significant variations in seed storage protein profiles among the genotypes and mutant generations. ‘Genotype 6’ showed a distinct 15.0kDa protein fragment which was absent in the majority of the remaining genotypes. The presence of distinct protein subunits in the three genotypes could be used in varietal / NRF

MSCAGR (Plant Production) / Department of Plant Production / Bambara groundnut (Vigna subterranea) is one of the most important legumes cultivated primarily for food by smallholder farmers in Africa. It is an affordable source of protein and contributes to income generation as well as soil fertility. Despite its importance, it is cultivated largely for subsistence purposes in South Africa. Growers use landraces. The agronomic performance of the traditional varieties depends on environmental factors prevailing in a particular area. In Limpopo and Mpumalanga Provinces, there is no adequate information regarding the performance of bambara groundnut germplasm. The objectives of the study were to (i) determine the agronomic performance of Bambara groundnut across three contrasting locations in Limpopo and Mpumalanga provinces over two cropping seasons (ii) determine the genotypic variation in the seed protein level among 42 bambara groundnut genotypes. Forty-two bambara groundnut genotypes were evaluated under three different environmental conditions (Syferkuil, Thohoyandou and Nelspruit) over two (2013/2014, 2014/2015) seasons in a 7 × 6 rectangular lattice design replicated three times. Eight agronomic traits including dry shoot weight (DSW), number of pods per plant (NPP), pod length (PL), number of seed per pod (NSP), pod weight per plant (PWT), seed weight per plant (SWT), 100 seed weight (100-SWT) and seed yield (SYLD) were measured. The results showed that there were significant genotype x location interactions which demonstrated that the prevailing agro-ecological conditions at the test locations were distinct from each other. Five genotypes (‘BGN-19‘, ‘BGN-11‘, ‘BGN-12‘, ‘BGN-4‘and ‘BGN-34‘) attained >25.0% seed yield advantage over the local check ‘BGN-39‘. The results also showed that light brown coloured genotypes attained relatively higher seed yield compared to the other seed colours types. The cultivar superiority index (CSI) showed that three genotypes (‘BGN-12‘, ‘BGN-19’ and ‘BGN-34’) were the most stable across the test locations and attained >900.0 kg/ha on average. There were significantly high positive correlations between PWT and each of the three other attributes (SWT, 100 SWT and SYLD). In terms of seed protein, the results showed a poor relationship between seed yield and protein levels. ‘BGN-12’ which produced the highest seed yield, attained the lowest percent seed protein while genotype. On average, the genotypes contained 21.72% protein. The highest and lowest seed protein quantities were attained by the genotypes ‘BGN-42’ (25.17%) and ‘BGN-12’ (19.89%) respectively. / NRF

No description available.