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.

An extensive investigation into and charactaisation of factors influencing transgene expression following introduction of the transgoie into tobacco via Agrobacterium- mediated transformation was carried out. Characterisation of material supplied at the outset of this project revealed that this material was unacceptable for further analysis. It was thus deemed necessary to obtain large populations of transgenic tobacco heterogenous for levels of transgene expression. Characterisation of these populations (CaMV-lecA and ssRubisco-lecA plants) showed that all plants fell into one of four segregation classes based on segregation of the kanamycin-resistance selectable marker. Results showed that the majority of regenerants contained multiple nptII-containing inserts, while the presence of one or two such inserts was also found, albeit at a much lower frequency. Segregation analysis based on detection of the lecA transgene agreed, in the majority of cases, with these results. However, in a few cases it was found that data obtained from both segregation analyses did not agree, with the presence of a single lecA-containing transgene being detected in plants shown to contain two copies of the nptII-contaning transgene. This result indicates the occurrence of T-DNA rearrangement either within the tobacco genome or during T-DNA transfer and integration. Southern blot analyses allowed a detailed characterisation of T-DNA structure, copy number and number of integration sites to be undertaken. Results from these analyses revealed a higher frequency of T-DNA rearrangement within plants containing multiple inserts. However, such rearrangements did not correlate with a significant reduction in levels of transgene expression since all detected rearrangements were found to occur at or towards the left hand border of the T-DNA, that border distant to the lecA transgene. Plants containing more than one T-DNA were also frequently found to contain these T-DNAs arranged as an inverted repeat at a single locus although no significant relationship between copy number and the presence of such structures was found. Correlating transgene expression levels, as determined by radioimmunoassay-based quantitation of lectin protein in tissues of transgenic plants, with T-DNA copy number, organisation and structure revealed no significant relationship. It is thus feasible to conclude that the major contributory factor influencing levels of transgene expression is the location of T-DNA integration within the plant genome. Subsequent work concerned with investigating the nature of those integration site-specific factors i.e. 'position effect' indicated a possible role for methylation-induced modulation of gene expression. Results presented in this thesis provide an insight into the fate of transgenes following introduction into the plant genome and clearly demonstrate the importance of further exploring the molecular mechanisms underlying transgene expression variability.

Thesis (M.S.)--Ohio University, August, 2008. / Title from PDF t.p. Includes bibliographical references.

The antibiotic, 2,4-diacetylphloroglucinol (Phl), is produced by a range of naturally isolated fluorescent pseudomonads, found in disease suppressive soils. The natural isolate, P. fluorescens F113, protects pea plants from the pathogenic fungus, Pythium ultimum, by reducing the number of pathogenic lesions on the plant's roots. This beneficial effect was however, outweighed by the F113 causing an overall reduction in the emergence of the pea plants in the infected soil. The gene locus responsible for the Phl production was shown to be functionally conserved between the P. fluorescens F113 and another Phl producing organism, P. fluorescens Q2-87. Following identification of this functional sequence homology, the genes were isolated from F113, by optimised, long PCR. The 6.7-kb gene cluster was inserted into the chromosome of a non-pathogenic P. fluorescens, SBW25, which can effect biological control against the plant pathogen, Pythium ultimum through competitive exclusion of the fungus, by means of its strong colonising competence. The insertion was a targeted, homologous recombination designed to insert the Phl coding genes, from the F113, into a non-essential, lacZY coding region of the SBW25 chromosome. The transformed strains of SBW25 assumed two different morphological appearances. The morphological changes were noted at a ratio of 1:1 of normal morphology and altered morphology. Transformation of SBW25 with the Phl locus without this repressor element led to transformants with only normal morphology. All transformants were able to suppress P. ultimum through antibiotic production following the Phl transformation. However, the fitness of the transformants was reduced in flask culture, at 30°C, against the un-transformed SBW25. The organisms transformed with the entire Phl locus were seen to clump together in the culture media. The strain transformed with the Phl locus lacking the repressor element behaved normally. When inoculated on pea seedlings, the strain containing no repressor element behaved similarly to the F113, causing lower pea seed emergence. A transformant containing the entire Phl genetic locus had not lost its environmental competence on the pea roots, maintaining a high population, but was unable to maintain a high population in the surrounding soil.

The purpose of this research problem is to determine the chemical composition of the blackeyed pea and to make a comparative study of the results. The value of the blackeyed pea as food, its chemical nature, and possible industrial uses are studied and recorded.

Peas are rich in protein and dietary fiber and can be used to create specialty products; however, flavor issues are one of the primary concerns regarding utilization. Sensory evaluations indicated the optimal treatment utilized aqueous ethanol at a concentration of 47.5%, extraction time of 63 min, and no pressure. Decreased (P<0.05) moisture and ash content, with no loss of protein or starch, were observed after treatment. Foaming properties were poor, indicating protein modification. Increased water absorption impacted WAI, WSI, setback, and peak time observations. Remaining pasting profile values were unchanged (P<0.05). While some volatiles were released via changes in protein and starch structure, total ppm decreased. Treated pea flour products had significantly (P<0.05) higher flavor acceptance scores. Texture results suggested treated flour imparted softness of baked items. Shelf-life measurements were improved for both cookies and crackers using treated pea flour.

Pigeonpea (Cajanus cajan (L.) Millsp.) is a drought tolerant legume of the Fabaceae family in the order Fabales and the only cultivated species in the genus Cajanus. It is mainly cultivated in the semi-arid tropics of Asia and Oceania, Africa and America. In Malawi, one of the top producers of pigeonpea in Africa, it is grown by small scale farmers as a source of food and income and for soil improvement in intercropping systems. However, varietal contamination due to natural outcrossing causes significant yield losses for farmers. In this study, 48 polymorphic SSR markers were used to assess diversity in all pigeonpea varieties cultivated in Malawi with the aim of developing a genetic fingerprint to distinguish the released varieties. SSR alleles were separated by capillary electrophoresis on an ABI 3700 automated sequencer and allele sizes determined using GeneMapper 4.0 software. Allelic data was analysed with PowerMarker. A total of 212 alleles were revealed averaging 5.58 alleles per marker with a maximum number of 14 alleles produced by CCttc019 (Marker 40). Polymorphic information content (PIC) ranged from 0.03 to 0.89 with an average of 0.30. DARwin software was used to generate a neighbour-joining tree that displayed three major clusters with two sub clusters in Cluster I. The released varieties were scattered across all the clusters observed, indicating that they generally represent the genetic diversity available in Malawi, although it was observed that there is substantial variation that can still be exploited through further breeding. Screening of the allelic data associated with five popular pigeonpea varieties for which a DNA fingerprint was to be developed, revealed 6 markers – CCB1 (Marker 1), CCB7 (Marker 2), Ccac035 (Marker 7), CCttc003 (Marker 15), Ccac026 (Marker 37) and CCttc019 (Marker 40)– which gave unique allelic profiles for each of the five varieties. With further tests needed for its robustness, this genetic fingerprint can be used for seed certification to ensure only genetically pure seeds are delivered to Malawi farmers. / Agriculture and  Animal Health / M. Sc. (Agriculture)

Field pea (Pisum sativum L.) as a cool season legume crop is sensitive to high day time temperature, especially during flowering. A population of 107 recombinant inbred lines (RILs) known as PR-11 was made from the cross of CDC Centennial (heat tolerant cultivar) X CDC Sage (heat sensitive cultivar) with the objectives of screening heat tolerant traits during flowering and subsequent seed development, and to map the quantitative trait loci (QTLs) responsible for these traits. Experiments were carried out in 2012-2014. PR-11 was seeded at normal seeding dates in 2012 and 2013 at Saskatoon (52º12’N, 106º63’W) and Rosthern (52º66’N, 106º33’W) in Canada, and in 2014 PR-11 was seeded at both normal and late seeding (three weeks later than normal) dates at one location, Saskatoon. Correlation analyses demonstrated that the duration of flowering (DOF) was positively associated with final seed yield under both normal and late seeding date conditions. Yield component traits on the main-stem [reproductive node number (Rnode), pod number (Pod), seed number per pod (Seed), single seed weight (SSW)] were significantly associated with main-stem seed yield, among which pod number appeared to be the component most positively associated with seed yield. However, yield on the main-stem was not significantly associated with seed yield at the plot level, which inferred that the contribution of seed yield on side branches was important. A genetic map consisting of 369 SNPs markers with a total coverage of 746 cM was developed using JoinMap 4.0. A total of 14 QTLs were detected under environments with normal seeding date, six for flowering traits, and eight for yield component traits. Eight QTLs were identified at late seeding, four for flowering traits and four for yield component traits. The total variation in days to flowering (DTF), DOF, Pod, Seed, SSW and grain yield that were each explained by the QTLs under normal seeding environments was 24 %, 43%, 15%, 32%, 34% and 21%, respectively. The QTLs together accounted for 43% of DTF variation, 14% of DOF variation, 17% of Pod variation, 12% of SSW variation and 12% of grain yield variation at the late seeding date. Lines PR-11-2, PR-11-88 and PR-11-91 performed as the top yielding lines under both normal and late seeding environments, and could be considered as heat tolerant lines.

No description available.

No description available.