Evaluation of the possible application of cowpea genotypes in the farming systems of the Eastern Cape Province, South Africa
- Authors: Adeyemi, Samson Adebowale
- Date: 2012
- Subjects: Cowpea -- South Africa -- Eastern Cape , Cowpea , Plant diversity , Cowpea -- Genetics , DNA fingerprinting of plants
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11274 , http://hdl.handle.net/10353/d1007539 , Cowpea -- South Africa -- Eastern Cape , Cowpea , Plant diversity , Cowpea -- Genetics , DNA fingerprinting of plants
- Description: Characterization studies on the genetic diversity among cultivated cowpea (Vigna unguiculata (L.) varieties are valuable tools to optimize the use of available genetic resources by farmers, local communities, researchers and breeders. Eight cowpea (Vigna unguiculata (L.) genotypes ( Vegetable cowpea, Ivory grey, Okhalweni, Fahari, Fahari dark, 97K-1069-8, IT93K-73h, and 129-3) were subjected to molecular, morphological and agronomical characterization. DNA amplification fingerprinting markers were used to evaluate the genetic diversity among the eight genotypes. Nine random arbitrary primers were used to screen the eight genotypes to assess their ability to reveal polymorphisms in cowpea, and seven of them were selected for use in characterizing the total sample. A total of 43 bands were generated which are all polymorphic. On the average, the primers generated a total of 6.1 polymorphic bands. The resulting data-matrix included 43 analysed bands with a total of 344 characters. Neighbour joining analysis was used to generate the dendrogram, clustering the genotypes into two groups at an agglomerate coefficient of 0.30 irrespective of their geographical origins. The results also showed the presence of significant differences in morphological and quality traits among the genotypes. Fahari yielded the highest concentration of crude protein (46.51 mg/mg dry leaf) while Vegetable cowpea yielded the lowest (24.41 mg/mg dry leaf). The influence of manure was also found to be effective by increasing the crude protein content of the genotypes as shown by Fahari dark with an average of 53.53 mg/mg dry leaf as opposed to 39.85 mg/mg dry leaf without manure application. Although some small clusters grouped accessions of the same growth habits, a general lack of agreement between clustering and morphological features was observed. It can therefore be concluded that the significant differences between the molecular genetic analysis using DAF-PCR markers, morphologic characters and yield traits can be important tools to identify and discriminates the different cowpea genotypes.
- Full Text:
- Authors: Adeyemi, Samson Adebowale
- Date: 2012
- Subjects: Cowpea -- South Africa -- Eastern Cape , Cowpea , Plant diversity , Cowpea -- Genetics , DNA fingerprinting of plants
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11274 , http://hdl.handle.net/10353/d1007539 , Cowpea -- South Africa -- Eastern Cape , Cowpea , Plant diversity , Cowpea -- Genetics , DNA fingerprinting of plants
- Description: Characterization studies on the genetic diversity among cultivated cowpea (Vigna unguiculata (L.) varieties are valuable tools to optimize the use of available genetic resources by farmers, local communities, researchers and breeders. Eight cowpea (Vigna unguiculata (L.) genotypes ( Vegetable cowpea, Ivory grey, Okhalweni, Fahari, Fahari dark, 97K-1069-8, IT93K-73h, and 129-3) were subjected to molecular, morphological and agronomical characterization. DNA amplification fingerprinting markers were used to evaluate the genetic diversity among the eight genotypes. Nine random arbitrary primers were used to screen the eight genotypes to assess their ability to reveal polymorphisms in cowpea, and seven of them were selected for use in characterizing the total sample. A total of 43 bands were generated which are all polymorphic. On the average, the primers generated a total of 6.1 polymorphic bands. The resulting data-matrix included 43 analysed bands with a total of 344 characters. Neighbour joining analysis was used to generate the dendrogram, clustering the genotypes into two groups at an agglomerate coefficient of 0.30 irrespective of their geographical origins. The results also showed the presence of significant differences in morphological and quality traits among the genotypes. Fahari yielded the highest concentration of crude protein (46.51 mg/mg dry leaf) while Vegetable cowpea yielded the lowest (24.41 mg/mg dry leaf). The influence of manure was also found to be effective by increasing the crude protein content of the genotypes as shown by Fahari dark with an average of 53.53 mg/mg dry leaf as opposed to 39.85 mg/mg dry leaf without manure application. Although some small clusters grouped accessions of the same growth habits, a general lack of agreement between clustering and morphological features was observed. It can therefore be concluded that the significant differences between the molecular genetic analysis using DAF-PCR markers, morphologic characters and yield traits can be important tools to identify and discriminates the different cowpea genotypes.
- Full Text:
Evaluation of various proteomic techniques to identify proteins involved in cereal stress responses to aphid infestation
- Authors: Nqumla, Ntombekhaya
- Date: 2012
- Subjects: Aphids , Wheat , Plant proteomics , Rice
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11270 , http://hdl.handle.net/10353/d1004572 , Aphids , Wheat , Plant proteomics , Rice
- Description: All plants are exposed to abiotic and biotic stresses and have developed intricate signalling responses to survive. They respond to cell-structure disruption caused by herbivore probing and feeding by the formation of callose. Callose is a linear homopolymer made up of β-1,3-linked glucose residues with some β-1,6-branches. Plant responses to abiotic or biotic stress share events such as phosphorylation, membrane depolarization, calcium influx and the release of reactive oxygen species such as hydrogen peroxide. These events lead to the up-regulation of several pathways leading to biosynthesis of signalling molecules such as salicylic acid, jasmonate, abscisic acid and ethylene pathways. The aim of this study was to determine the most suitable proteomic approach for identifying proteins and signalling pathways involved in cereal response to aphid infestation. An in silico approach was first evaluated in which the 5ʹ upstream regulatory region of proteins belonging to the family of callose synthases was scanned for cis-regulatory elements in order to identify which callose synthases are possibly expressed in plants during biotic or abiotic stresses. Bioinformatics tools were used in the identification of twelve Arabidopsis and ten rice callose synthase coding regions. Genome sequences for rice and Arabidopsis were scanned for the 2000 bp 5ʹ region upstream of the start codon of each callose synthase coding region. PlantCare, PLACE and Athena software were used to identify putative cis-regulatory elements present in the 2000 bp 5ʹ upstream sequences. The majority of cis-acting elements identified were involved in drought and high temperature responses and only one cis-acting element was involved in wound stress. These results therefore indicated a probable role for plant callose synthases in drought stress responses rather than in biotic stress responses. Genevestigator analysis of Arabidopsis results of micro-array experiments indicated that AtGSL10 is highly up-regulated, with AtGSL1, 3, 5, 6, 7, 8, 11 and 12 showing medium up-regulation and AtGSL2, 4 and 9 no up-regulation during aphid infestation of Arabidopsis plants, implicating a possible role for AtGSL10 in the plant response to aphid infestation. An LC/MS/MS approach was used to identify specific signalling pathways involved in wheat resistance or stress response to aphid infestation. Eight proteins were identified as being up-regulated during aphid feeding in wheat, and 11 proteins were identified as possibly involved in the wheat resistance mechanism against aphid infestation. Several proteins were also identified as constitutively expressed proteins, during normal conditions and aphid infestation. Most pathways identified with proteins up-regulated in the resistance mechanisms of TugelaDN plants, were related to energy metabolism and located in the chloroplast. Evaluation of two dimensional gel electrophoresis to identify phosphoproteins differentially regulated in wheat during aphid infestation, revealed the up-regulation of three proteins namely photosystem II oxygen-evolving complex protein 2, HVUNKNOWN from Hordeum vulgare subsp vulgare and HSKERAT9 NID from Homo sapiens. Additional 57 proteins were partially identified as involved in the stress response but due to low protein levels, the percentage of matching peptides to these proteins was below the required confidence level. Although these protein identifications were below the confidence level, it is interesting to note that several of the proteins are known stress response proteins, and therefore could serve as potential targets for future investigations. In conclusion, the down and up-regulation of wheat proteins after aphid feeding reported in this study suggest that several signalling pathways are involved in the cereal stress response to aphid feeding. In silico approaches require knowledge or identification of potential proteins whereas 2D and LC/MS can identify numerous proteins still unknown to be involved in specific stress responses. The 2D approach is also limited in that the proteins of interest may be in low abundance and therefore not detected in the gels due to the presence of high abundant proteins. Therefore the best approach to identify proteins and signalling pathways involved in the stress response of wheat to aphid infestation, is the LC/MS/MS approach, as this proved to be the most sensitive and robust, identifying the most proteins with a high degree of confidence.
- Full Text:
- Authors: Nqumla, Ntombekhaya
- Date: 2012
- Subjects: Aphids , Wheat , Plant proteomics , Rice
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11270 , http://hdl.handle.net/10353/d1004572 , Aphids , Wheat , Plant proteomics , Rice
- Description: All plants are exposed to abiotic and biotic stresses and have developed intricate signalling responses to survive. They respond to cell-structure disruption caused by herbivore probing and feeding by the formation of callose. Callose is a linear homopolymer made up of β-1,3-linked glucose residues with some β-1,6-branches. Plant responses to abiotic or biotic stress share events such as phosphorylation, membrane depolarization, calcium influx and the release of reactive oxygen species such as hydrogen peroxide. These events lead to the up-regulation of several pathways leading to biosynthesis of signalling molecules such as salicylic acid, jasmonate, abscisic acid and ethylene pathways. The aim of this study was to determine the most suitable proteomic approach for identifying proteins and signalling pathways involved in cereal response to aphid infestation. An in silico approach was first evaluated in which the 5ʹ upstream regulatory region of proteins belonging to the family of callose synthases was scanned for cis-regulatory elements in order to identify which callose synthases are possibly expressed in plants during biotic or abiotic stresses. Bioinformatics tools were used in the identification of twelve Arabidopsis and ten rice callose synthase coding regions. Genome sequences for rice and Arabidopsis were scanned for the 2000 bp 5ʹ region upstream of the start codon of each callose synthase coding region. PlantCare, PLACE and Athena software were used to identify putative cis-regulatory elements present in the 2000 bp 5ʹ upstream sequences. The majority of cis-acting elements identified were involved in drought and high temperature responses and only one cis-acting element was involved in wound stress. These results therefore indicated a probable role for plant callose synthases in drought stress responses rather than in biotic stress responses. Genevestigator analysis of Arabidopsis results of micro-array experiments indicated that AtGSL10 is highly up-regulated, with AtGSL1, 3, 5, 6, 7, 8, 11 and 12 showing medium up-regulation and AtGSL2, 4 and 9 no up-regulation during aphid infestation of Arabidopsis plants, implicating a possible role for AtGSL10 in the plant response to aphid infestation. An LC/MS/MS approach was used to identify specific signalling pathways involved in wheat resistance or stress response to aphid infestation. Eight proteins were identified as being up-regulated during aphid feeding in wheat, and 11 proteins were identified as possibly involved in the wheat resistance mechanism against aphid infestation. Several proteins were also identified as constitutively expressed proteins, during normal conditions and aphid infestation. Most pathways identified with proteins up-regulated in the resistance mechanisms of TugelaDN plants, were related to energy metabolism and located in the chloroplast. Evaluation of two dimensional gel electrophoresis to identify phosphoproteins differentially regulated in wheat during aphid infestation, revealed the up-regulation of three proteins namely photosystem II oxygen-evolving complex protein 2, HVUNKNOWN from Hordeum vulgare subsp vulgare and HSKERAT9 NID from Homo sapiens. Additional 57 proteins were partially identified as involved in the stress response but due to low protein levels, the percentage of matching peptides to these proteins was below the required confidence level. Although these protein identifications were below the confidence level, it is interesting to note that several of the proteins are known stress response proteins, and therefore could serve as potential targets for future investigations. In conclusion, the down and up-regulation of wheat proteins after aphid feeding reported in this study suggest that several signalling pathways are involved in the cereal stress response to aphid feeding. In silico approaches require knowledge or identification of potential proteins whereas 2D and LC/MS can identify numerous proteins still unknown to be involved in specific stress responses. The 2D approach is also limited in that the proteins of interest may be in low abundance and therefore not detected in the gels due to the presence of high abundant proteins. Therefore the best approach to identify proteins and signalling pathways involved in the stress response of wheat to aphid infestation, is the LC/MS/MS approach, as this proved to be the most sensitive and robust, identifying the most proteins with a high degree of confidence.
- Full Text:
Inhibitory potential of honey on the enzymatic activity of Helicobacter pylori urease
- Authors: Matongo, Fredrick
- Date: 2012
- Subjects: Honey , Helicobacter pylori infections , Enzyme inhibitors , Traditional medicine , Antifungal agents
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11253 , http://hdl.handle.net/10353/431 , Honey , Helicobacter pylori infections , Enzyme inhibitors , Traditional medicine , Antifungal agents
- Description: Urease of Helicobacter pylori is an important virulence factor implicated in the pathogenesis of many clinical conditions, such as chronic gastritis, peptic ulceration, and gastric cancer. Many urease inhibitors have been discovered, like phosphorodiamidates, hydroxamic acid derivatives, and imidazoles. Despite good activities at the enzyme level and excellent kinetic properties most of them have not been used as therapeutic agents in vivo because of their side effects, toxicity and instability. This has led to much attention to focus on exploring the novel urease inhibitory activities of natural products because of their low toxicity and good bioavailability. Honey, a natural product has been used in folk medicine due to its antitumor, antioxidant, antimicrobial and anti-inflammatory properties. The aims of this study were to isolate, characterise, purify urease produced by H. pylori and investigate the inhibitory effects of solvent honey extracts on its enzymatic activity. Urease was found to be both surface-associated and cytoplasmic. Maximum cytoplasmic urease activity was found to occur after 72 hr whereas maximum extracellular urease activities were found to occur after 96 hr. Characterization of the crude cytoplasmic urease revealed optimal activity at a pH of 7.5 and temperature of 40°C. The kinetic parameters Vmax and Km were 45.32 U ml-1 and 61.11 mM respectively.The honey extracts inhibited the activity of the crude urease in a concentration dependent manner. The Lineweaver-Burk plots indicated a non-competitive type of inhibition against H. pylori urease. The two honey extracts gave promising inhibitory activities against urease of H. pylori. Thus the results of this study delineates that inhibition of urease can ease development in therapeutic and preventative approaches based on the enzymatic activity of this Helicobacter protein.
- Full Text:
- Authors: Matongo, Fredrick
- Date: 2012
- Subjects: Honey , Helicobacter pylori infections , Enzyme inhibitors , Traditional medicine , Antifungal agents
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11253 , http://hdl.handle.net/10353/431 , Honey , Helicobacter pylori infections , Enzyme inhibitors , Traditional medicine , Antifungal agents
- Description: Urease of Helicobacter pylori is an important virulence factor implicated in the pathogenesis of many clinical conditions, such as chronic gastritis, peptic ulceration, and gastric cancer. Many urease inhibitors have been discovered, like phosphorodiamidates, hydroxamic acid derivatives, and imidazoles. Despite good activities at the enzyme level and excellent kinetic properties most of them have not been used as therapeutic agents in vivo because of their side effects, toxicity and instability. This has led to much attention to focus on exploring the novel urease inhibitory activities of natural products because of their low toxicity and good bioavailability. Honey, a natural product has been used in folk medicine due to its antitumor, antioxidant, antimicrobial and anti-inflammatory properties. The aims of this study were to isolate, characterise, purify urease produced by H. pylori and investigate the inhibitory effects of solvent honey extracts on its enzymatic activity. Urease was found to be both surface-associated and cytoplasmic. Maximum cytoplasmic urease activity was found to occur after 72 hr whereas maximum extracellular urease activities were found to occur after 96 hr. Characterization of the crude cytoplasmic urease revealed optimal activity at a pH of 7.5 and temperature of 40°C. The kinetic parameters Vmax and Km were 45.32 U ml-1 and 61.11 mM respectively.The honey extracts inhibited the activity of the crude urease in a concentration dependent manner. The Lineweaver-Burk plots indicated a non-competitive type of inhibition against H. pylori urease. The two honey extracts gave promising inhibitory activities against urease of H. pylori. Thus the results of this study delineates that inhibition of urease can ease development in therapeutic and preventative approaches based on the enzymatic activity of this Helicobacter protein.
- Full Text:
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