Peroxiredoxins are a family of multifunctional enzymes that are able to protect the cell against oxidative stress. Peroxiredoxins form part of a recently discovered peroxide scavenging system along with thioredoxin, thioredoxin reductase and sulfiredoxin. This study describes the purification of a recombinant human peroxiredoxin II from human erythrocytes. The original recombinant clone contained a point mutation at the fourth residue from glycine to valine and a number of problems were encountered with aggregation during purification. Reverting back to the original amino acid sequence allowed the protein to be purified and concentrated without aggregation, as well as leading to over-expression in the same oligomeric state as the native sample from blood. This study also describes the over-expression and purification of the human peroxiredoxin II protein in the intermolecular disulfide form as well as the subsequent crystallisation and X-ray diffraction studies. The crystal structure for this form of the protein was obtained to 3.3 Å resolution revealing the peroxiredoxin to be in the decameric form. In addition conformational changes in the protein that are necessary for formation of the intermolecular disulfide between the peroxidatic (Cys52) and resolving cysteine (Cys172) have been observed. The structure also revealed that these movements did not interfere with the dimer:dimer interface as had been previously suggested. This then allows the disulfide to be seen within the decameric form of peroxiredoxin. The production of covalent complexes formed between peroxiredoxin and sulfiredoxin, and peroxiredoxin and thioredoxin was also investigated. Complexes were stabilised by using DTNB to form a covalent bond between specific cysteine residues. The complex binding results from size exclusion chromatography showed that decameric peroxiredoxin bound to sulfiredoxin in a 1:5 ratio and decameric peroxiredoxin bound to thioredoxin in a 1:10 ratio. Cloning, over-expression and purification of the selenocysteine containing enzyme thioredoxin reductase was achieved. A minimal selenocysteine insertion sequence was added to the 3’ end of the DNA sequence to drive selenocysteine insertion in place of the typical stop UGA codon. The activity of this protein was found to be low but was greatly increased when co-expressed with a plasmid containing the selA, selB and selC genes. Although the activity of this co-expressed thioredoxin reductase was ~20% of the native enzyme activity, it was comparable to the activity of other recombinant forms of the enzyme. These studies report the purification of all of the proteins necessary to reform the peroxiredoxin system and allow the production of a working assay for peroxiredoxin activity. Together with the first report for a structure of a decameric disulfide form of human peroxiredoxin II a greater insight into the peroxiredoxin system has been obtained.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.