Genetics - Theses
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ItemNovel roles of cytochrome P450 genes in insect development(University of Melbourne, 2009)Cytochrome P450s form one of the largest enzyme superfamilies. They are involved in the catalysis of a number of different chemical reactions and are involved in a wide range of biological processes. In the vinegar fly, Drosophila melanogaster, 85 P450s have been identified. This thesis uses genetic approaches to investigate the roles of specific P450s in different aspects of D. melanogaster development and reproduction. The expression patterns of all 85 P450s in the D. melanogaster y ; cn, bw; sp strain were analysed by RT-PCR and in situ hybridisation during embryogenesis. In situ hybridisation patterns were obtained from 50 out of 65 P450s expressed during embryogenesis. Many novel expression patterns were identified; Cyp28c1 was detected in the salivary glands, Cyp49a1 was detected in the hindgut and Cyp18a1 was detected in the epidermis. These results were compared with larval in situ hybridisation patterns of P450s previously characterised, to identify P450s with conserved expression throughout the life-stage. An RNAi screen was also performed. Of the 60 P450s targeted by RNAi, nine were shown to be lethal (Cyp18a1, Cyp28c1, Cyp306a1, Cyp309a1, Cyp311a1, Cyp314a1, Cyp4c3, Cyp4d2, Cyp4g1, Cyp6g2) and three showed reduced survival (Cyp318a1, Cyp4ac2, Cyp4s3). The function of Cyp301a1 was also investigated using in situ hybridisation and RNAi. Cyp301a1 was detected in the hindgut and epidermis in D. melanogaster y; cn, bw; sp embryonic and adult stages. RNAi knockdown of Cyp301a1 as well as analysis of the Cyp301a1f02301 mutant flies showed disrupted cuticle phenotypes, suggesting that Cyp301a1 has a role in cuticle formation. Cyp6g2 was the only P450 detected in the corpus allatum, the site of juvenile hormone synthesis. RNAi knockdown of Cyp6g2 is lethal at pupal stages and lethality can be rescued by over-expression of D. willistoni Cyp6g2. Cyp6g2 appears to be unique to the Drosophila genus and is highly conserved in all twelve sequenced Drosophila genomes. Cyp6g2 expression fluctuates dynamically during larval and pupal development and is roughly correlated with high JH titres. Knockdown of Cyp6g2 disrupts the expression of other genes known to be regulated by JH, suggesting that Cyp6g2 may have an essential role in juvenile hormone regulation. Interestingly, Cyp6g2 is also expressed in the adult male ejaculatory bulb and was differentially expressed during mating, indicating an additional reproductive role. The evolution of the Cyp307a genes in Drosophila was investigated. The Cyp307a genes are involved the synthesis of 20-hydroxyecdysone, an essential insect moulting hormone. In D. melanogaster, there are two Cyp307a genes; Cyp307a1, which was detected in the yolk cells of the early embryo and in the follicle cells of the female ovary and Cyp307a2, which was detected in the prothoracic cells of the ring gland. Phylogenetic and microsynteny analyses in Drosophila showed a complicated evolutionary scenario for the Cyp307a genes, invoking multiple gene duplications (and subsequent losses) in different lineages. The varied temporal and spatial expression of the Cyp307a genes in Drosophila suggested that they have undergone independent evolution and sub-functionalisation within the Drosophila genus. Finally, this thesis investigated roles of cytochrome P450s in reproduction. Using in situ hybridisation, several P450s were detected in the adult female reproductive tissues. Cyp6a19 and Cyp6a22 were highly expressed in the nurse cells whereas Cyp18a1 was detected in the follicle cells of the developing oocyte. Cyp305a1 was detected in the follicle cells covering the late stage oocyte and in the dorsal region specifying the appendage. Ubiquitous knockdown of Cyp305a1 by RNAi was lethal, suggesting it plays an essential role during development. RNAi of Cyp305a1 in the follicle cells, showed defects in nurse cell dumping and dorsal appendage formation. Several P450s were also detected in the adult male reproductive system, with Cyp312a1 expressed highly in the developing spermatocytes of the male testes. RNAi knockdown of Cyp312a1 was viable and Cyp312a1 RNAi progeny showed normal testes development. However Cyp312a1 RNAi males were less fertile, which may be due to defects in sperm maturation or protection in the female reproductive tract.