Browsing Doctoral Degrees (Genetics) by Author "Carson, Deborah L. (Deborah Lee)"
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- ItemGene discovery and expression analysis in sugarcane leaf and culm(Stellenbosch : Stellenbosch University, 2002-12) Carson, Deborah L. (Deborah Lee); Botha, F. C.; Huckett, B.; Stellenbosch University. Faculty of AcriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Sugarcane (Saccharum spp. hybrids) is a commercial crop plant capable of storing up to 20% sucrose on a fresh mass basis in the culm. Knowledge about gene expression during sugarcane growth and maturation is limited. The aim of this study was to assess whether an Expressed Sequence Tag (EST)-based approach towards analysis of sugarcane would reveal new information about gene expression and metabolic processes associated with sugarcane growth and development. The specific objectives were two-fold: firstly, to develop an EST database for sugarcane and secondly, to identify and analyse genes that are expressed in different sugarcane tissue types and developmental stages, with a specific focus on leaf and culm. An EST database for sugarcane was initiated to obtain information on sugarcane gene sequences. A total cDNA library was constructed from sugarcane immature leaf (leaf roll: meristematic region) tissue and 250 clones randomly selected and subjected to single-pass DNA sequence analysis. Sugarcane ESTs were identified by sequence similarity searches against gene sequences in international databases. Of the 250 leaf roll clones, 26% exhibited similarity to known plant genes, 50% to non-plant genes while 24% represented new gene sequences. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes. A significant proportion of genes identified in the leaf roll were involved in processes related to protein synthesis and protein modification, as would be expected in meristematic tissues. Submission of 495 sugarcane gene sequences to the dbEST database represented the first sugarcane ESTs released into the public domain. Two subtracted cDNA libraries were constructed by reciprocal subtractive hybridisation between sugarcane immature and maturing internodal tissue. To explore gene expression during sugarcane culm maturation, partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries was performed. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases while 111 matched uncharacterised plant ESTs only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. The expression patterns of sugarcane genes were examined in different tissue sources and developmental stages to identify differentially expressed genes. cDNA arrays containing 1000 random clones from immature leaf and maturing culm cDNA libraries were hybridised with poly (At RNA from immature leaf, mature leaf, immature culm and maturing culm. All cDNAs examined hybridised to all four probes, but differences in signal intensity were observed for individual cDNAs between hybridisation events. No cDNAs displaying tissue- or developmental-stage specific expression were detected. Comparisons between hybridisation patterns identified 61 cDNAs that were more abundantly expressed in immature and mature leaf than the culm. Likewise, 25 cDNAs preferentially expressed in immature and maturing culm were detected. ESTs established for the differentially expressed cDNAs revealed sequence homology to a diverse collection of genes in both the leaf and the culm. These included genes associated with general cellular metabolism, transport, regulation and a variety of stress responses. None of the differentially expressed genes identified in the culm were homologous to genes known to be associated with sucrose accumulation. To examme differences at the level of gene transcription between low sucroseaccumulating and high sucrose-accumulating tissues, subtracted cDNA libraries were utilised. To isolate cDNAs differentially expressed during culm maturation, cDNA arrays containing 400 random clones (200 from each library) were screened with total cDNA probes prepared from immature and maturing culm poly (At RNA. Results indicated that 36% and 30% of the total number of cDNAs analysed were preferentially expressed in the immature and maturing culm, respectively. Northern analysis of selected clones confirmed culm developmental stage-preferential expression for most of the clones tested. ESTs generated for the 132 differentially expressed clones isolated exhibited homology to genes associated with cell wall metabolism, carbohydrate metabolism, stress responses and regulation, where the specific ESTs identified in the immature and maturing culm were distinct from each other. No developmentally regulated ESTs directly associated with sucrose metabolism were detected. These results suggest that growth and maturation of the sugarcane culm is associated with the expression of genes for a wide variety of metabolic processes. In addition, genes encoding enzymes directly involved with sucrose accumulation do not appear to be abundantly expressed in the culm.