Drosophila is a valuable experimental organism can be used as a reverse genetics model. Drosophila Malpighian (renal) tubules are important epithelial tissue in which to study transport mechanisms. RNA-seq has been chosen to investigate Drosophila Malpighian (renal) tubules to identify novel genes following a three- way comparison between three popular transcriptome profiling methods. Two types of novel gene have been found in Drosophila tubules, coding genes and noncoding genes. Reverse genetics has been applied to identify novel coding gene function in Drosophila tubules. Three-way analysis of Drosophila expression microarrays, Drosophila tiling micrarrays and Drosophila RNA-seq reveal that most gene expression levels are well correlated between the three technologies. Drosophila expression microarrays and RNA-seq are correlated better than the correlation between Drosophila tiling microarrays and RNA-seq. Drosophila expression arrays and Drosophila tiling arrays all suffered from cross-hybridization, miss target detection and hybridization background noise, and also have low dynamic range for detecting lowly and highly expressed genes. Drosophila tiling microarrays also have a high false-positive detection rate, which may lead to overestimate the transcriptional activities of the genome. RNA-seq has overcome the drawbacks of microarrays and become the leading technology for genome sequencing, transcriptome profiling, novel gene discovery, and novel alternative splicing discovery with wide dynamic range. However, Drosophila expression microarrays and tiling microarrays still remain useful. Three-prime expression microarrays offer a means to measure the differential three-prime end processing, and tiling microarrays can be used for novel gene discovery. In this sense, the three technologies complement each other. Poly(A) selected RNA-seq has been used as a discovery tool for searching novel genes in Drosophila Malpighian tubules in this thesis. A TopHat and Cufflinks pipeline has been used as an analytical pipeline for novel gene discovery and differential gene expression analysis between Drosophila tubules and whole flies in order to find the tubule-enriched genes. Reverse genetics has been applied to Drosophila to achieve a gene knockdown and overexpression by using the unique Gal4/UAS system to achieve the novel gene knockdown or overexpression in specific tissue and cell types. Novel coding gene CG43968 has been discovered. The location of this gene has been confirmed in tubule main segments, principle cell cytoplasm or apical membrane. The function of this gene has been identified as involvement in tubule secretion, which may relate to calcium transport. Reverse genetics has been confirmed as particularly important for the functional study of novel genes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:622024 |
| Date | January 2014 |
| Creators | Wang, Jing |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/5459/ |
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