Document Type Master's Dissertation Author Ranik, Martin URN etd-08042008-145138 Document Title Expression profiling and characterization of wood formation genes in Eucalyptus Degree MSc Department Genetics Supervisor
Advisor Name Title Dr A A Myburg Supervisor Keywords
- wood formation genes
Date 2005-09-07 Availability restricted AbstractEucalyptus trees are capable of generating vast amounts of wood which is a product of the highly structured differentiation of cell layers centred around the vascular cambium. In order to understand xylogenesis (wood formation), it is necessary that the genes which underlie this complex process be isolated and characterised. Previously, very little was known about the types of genes that are expressed in the main woody tissue layers during xylogenesis in the Eucalyptus stem. The aim of this study was to develop a high-throughput transcript profiling platform and to use it in generating a global view of the woody stem transcriptome. Additionally, it was ventured that the newly established system be used for the isolation of gene fragments expressed during wood development and culminate in the characterisation of novel genes important in wood formation.
High-throughput cDNA-AFLP expression profiling utilizing a combination of infrared fragment detection and semi-automated fragment quantification was used to profile the expression of 6385 transcript derived fragments (TDFs) in four major woody tissues (immature xylem, mature xylem, phloem and cork). Clustering of gene expression data revealed that approximately a fifth of the TDFs were differentially expressed and several clusters of TDFs with shared tissue-specific expression profiles were identified. Gene expression data derived from cDNA-AFLP band quantification was confirmed by quantitative RT-PCR. TDFs representing specific clusters were isolated, sequenced and assigned putative identities. A significant proportion of the 71 sequenced TDFs was found to be similar to genes known to play roles in processes associated with wood development – cell wall biosynthesis, cell fate and gene regulation. The technique of cDNA-AFLP performed using automated DNA analysers was found to be a powerful, fast and relatively inexpensive system for the analysis of gene expression coupled to gene discovery in forest trees. By quantifying and clustering TDFs with shared expression profiles, it was possible to isolate cDNA fragments of candidate genes for further characterization without the requirement for excessive sequencing.
One of the isolated TDFs, which was strongly upregulated in xylem, exhibited significant homology to cellulose synthase catalytic subunit genes (CesA) that are instrumental in the deposition of cellulose in the walls of all plant cells. In plants these genes comprise a small gene family of dissimilar members with distinct functions. Using degenerate RT-PCR as well as 5’ and 3’ RACE, six new full-length Eucalyptus grandis CesA cDNAs (EgCesA1 through 6) were isolated. The EgCesA cDNAs all span over 3.3kb and are predicted to encode proteins of 978 to 1097 amino acid residues which possess all of the motifs characteristic of functional CesAs. Sequence analysis and phylogenetic comparison to each other and to all currently known full-length plant CesA sequences suggested that EgCesA1 to 6 represent distinct family members rather than allelic variants or paralogs. Expression profiling of the six EgCesAs using quantitative RT-PCR revealed that EgCesA1 to 3 were very strongly expressed in tissues enriched for cells depositing secondary cell walls. By contrast, EgCesA4 and EgCesA were upregulated in tissues that contain mostly actively dividing cells, which are depositing primary walls, but include few cells that are undergoing secondary cell wall biogenesis. EgCesA6 was found to be expressed at very low levels in all tissues assayed. The six isolated EgCesAs represent a significant proportion of the Eucalyptus CesA gene family and are the first cellulose synthase catalytic subunit genes from Eucalyptus to be comprehensively characterised. The non-isotopic cDNA-AFLP approach optimised during this study can now be applied to more specific questions pertaining to gene expression profiling in trees. With further characterisation, the six newly isolated EgCesA genes will significantly contribute towards the understanding of cellulose biosynthesis in forest trees.
© University of Pretoria 2005
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