| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 96 === Abstract
The Cytochrome P450Ⅰ(CYPⅠ) is a subfamily of cytochrome P450 superfamily. It includes CYP1A1, CYP1A2 and CYP1B1, that are all regulated through aryl hydrocarbon receptor (AHR)-related signaling pathway. The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) protein family. AHR regulates downstream gene expression following activation by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) or a variety of other synthetic and natural compounds.
There are four cyp1 genes in zebrafish, including cyp1a1, cyp1b1, cyp1c1 and cyp1c2, which are all controlled by AHR signaling pathway. Cyp1a1 is mainly expressed in blood vessel, intestinal tract, swim bladder and eye. A basal level of cyp1b1 transcript is mainly expressed in the eye. TCDD exposure may induce a strong level of cyp1a1 transcription in a broad range of tissues, but only causes a mild level of cyp1b1 induction in branchial arches. To investigate the regulatory mechanism of cyp1b1 transcription, here I have constructed pCYP1B1 SP-EGFP and pCYP1B1 LP-EGFP reporter vectors, that contain 1619 bp and 3013 bp of cyp1b1 ORF upstream sequence, respectively. The recombinant vectors were microinjected into embryos at 1 cell-stage. The fluorescence of recombinant EGFP was not observed in both vectors. Nevertheless, the recombinant EGFP mRNA can be detected in eye, head and trunk tissues during developing stage by whole mount in situ hybridization analysis. There was no distinct reporter activity between the two constructs. Although there are three xenobiotics response elements (XRE) in the 1619 bp upstream of cyp1b1 ORF region, the recombinant vectors did not response to TCDD exposure significantly.
I have also examined function of AHR1B and AHR2 in cyp1b1 transcription during developing stage. Blockage of AHR1B translation alone or blockage of AHR1B and AHR2 together reduced the basal level of cyp1b1 transcription in retina region at 24 hpf, but not at 48 hpf. It suggests that AHR1B and AHR2 signaling pathways have important role in cyp1b1 transcription in early stage of development but do not involve in cyp1b1 transcription at late stage.
The correlation of ahr1a, ahr1b and ahr2 in zebrafish embryo was studied by gene-specific morpholino knockdown analysis. It appears that ahr1b is the only ahr gene that is controlled by all three AHR factors and its expression was inhibited by individual ahr knockdown. The rest two AHR genes, ahr1a and ahr2, were not influenced by any ahr knockdown. My results suggest that both zebrafish ahr1b and mammalian ahr have a similar positive feedback pathway.
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