Sorghum is a valuable grain crop and a principle source of food of particular importance in human and animal nutrition in the semi-arid regions of Africa and Asia. Despite its value, sorghum grain quality is a major limitation to its productivity and profitability. Sorghum grain is usually discounted as feed grain when compared to wheat and barley, predominantly because of its poor digestibility. The sorghum endosperm is composed of a complex starch protein matrix, whereby the starch is physically bound within the storage proteins, the kafirins. The kafirins are synthesised on the membrane bound polysomes and have a signal peptide which targets them to the lumen of the endoplasmic reticulum. Growth of protein bodies occur as - kafirins fill in the interior with and γ kafirins occupy the periphery. Despite the -kafirins being more digestible and composing of 60- 80% of the kafirins, they are not easily accessible due to and γ kafirins which have a high content of intermolecular disulphide bonds (S-S), rendering them highly resistant to proteases. Alteration of the structure of the protein bodies and change of the location of the-kafirins could result in a higher digestibility of sorghum proteins. This could be achieved by upregulating or downregulating the -kafirins. The improvement of grain quality, both in increased protein and starch digestibility would substantially enhance the digestibility of sorghum as animal feed as well as for human consumption. Various techniques have been utilised to classify the kafirins according to their mobility on SDS PAGE electrophoresis, Reverse Phase High Performance Liquid Chromatography (RP-HPLC), Free Zone Capillary Electrophoresis (FZCE) and Lab on Chip. Until recently the characterisation and classification of the kafirins generally have relied on the characterisation of zeins from maize. Zeins have about 70% homology to the kafirins both at the nucleotide and amino acid level. Based on the high similarity of the -kafirins to the -zeins, the - kafirins have been classified as 19 and 22 kDa. Despite their 70% homology the migration of the - kafirins on SDS PAGE is quite different to that of the zeins. Hence, I propose a new classification of the -kafirins as 23 kDa and 25 kDa based on their mobility on SDS PAGE Characterisation and cloning of the 23 and 25 kDa genes was performed using QL41 the Queensland inbred line of sorghum. Ten positive clones were isolated from a cDNA library for the 25 kDa and two clones for the 23 kDa -kafirins. The isolated clones of the 25 kDa -kafirins showed 98-99% homology with each other and also with the GenBank sequences. The major finding was the characterisation of the 23 kDa -kafirins. The two clones obtained showed 100% homology to each other as well as to the published sequences on the GenBank, and were full-length sequences. Also a partial sequence was obtained that lacked the signal peptide and was different to the other two clones. Whilst characterising the 23 kDa a second group of the 25 kDa -kafirins was identified from the genomic DNA, of all the three genotypes (QL41, 296B and QL12), which was unique from the previously isolated clones. This group of -kafirins was not among the cluster but was 5’ upstream of the cluster. This group had a higher content of the glutamine compared with the other 25 kDa group. The expression level was studied to show how each gene family contributed to the level of - kafirins. QL41 and 296B were used for this study. From the studies it was shown that the 23 kDa - kafirins genes were 20% more expressed than the 25 kDa. An attempt to identify suitable markers for the -kafirins was investigated using RFLP and SSR analysis. Thirty-two different genotypes were utilised for this study. The observed variation indicated by cluster analysis (4-38%) clearly showed variation of the -kafirins in genotype and within the kafirin genes as elucidated by the sequences in Chapter 4. Markers able to identify this variation could help in the selection of highly digestible mutants. Hence, there is potential for sorghum grain improvement using marker-assisted breeding. The need to identify a tissue specific promoter was essential, especially for a strong promoter that could drive expression in the endosperm of the monocots. A vector construct consisting of the - kafirin promoter driving the GUS reporter genes was used for transient expression from QL41. This was assessed in the sorghum and barley calli, sorghum endosperm and leaves and corn endosperm. Tissue specific expression as well as higher levels of transient expression were seen using the - kafirin promoter, compared with the ubiquitin promoter. Preliminary experiments have illustrated the potential use of a gene silencing mechanism that could enhance the digestibility of sorghum grain. The 25 kDa -kafirin gene was used as the target for gene silencing using the mechanism of iRNA. Transformation constructs were developed using the throughput vector pSTARGATE in an effort to silence the 25 kDa -kafirins. The characterisation of the -kafirins has provided valuable information for future sorghum improvement research.
| Identifer | oai:union.ndltd.org:ADTP/254236 |
| Creators | Pratibala Pandit |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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