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Alternative TitleExpression pattern and promoter function analysis of ovarian aromatase in red-spotted grouper Epinephelus akaara
Thesis Advisor桂建芳
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword卵型芳香化酶基因 赤点石斑鱼 启动子 性反转
Abstract本研究在实验室前期工作的基础上,从赤点石斑鱼卵巢SMART cDNA文库中克隆了卵型芳香化酶基因(EaCyp19a1a),并制备了特异性多克隆抗体,对其在成鱼体内的表达特征和调控机制进行了研究。 研究发现,EaCyp19a1a基因具有芳香化酶共有的保守区域,在进化中高度保守。其序列与鱼类卵型芳香化酶之间的同源性较高,并且存在一些特异保守的氨基酸残基。 实验检测发现,EaCyp19a1a在赤点石斑鱼成体卵巢组织中特异表达,且仅表达于卵巢滤泡细胞中。在人工性反转过程中,EaCyp19a1a表达量随着性腺由雌到雄的转变逐渐下降,在雄性精巢组织中检测不到表达。 克隆了EaCyp19a1a基因的启动子区域。序列分析发现,其上有包括SF-1、CREB、SOX5等在内的27个潜在转录因子结合位点。体外活性分析实验表明,在-1010到-898片段中的CREB转录因子对其启动子活性增强具有非常重要的顺势调控作用。显微注射实验发现EaCyp19a1a启动子能够指导绿色荧光蛋白在斑马鱼早期性腺中特异表达,这表明其序列中含有调控性腺特异表达的顺势作用元件,对EaCyp19a1a卵巢特异表达有调控作用。 同时,本研究还发现部分硬骨鱼类Cyp19a1a基因启动子近端有一段长约300bp的高度保守区域,部分转录因子结合位点如TATA框、SF-1、SOX5和CREB等也在该区域保守存在,推测该区域对鱼类Cyp19a1a卵巢特异表达具有重要意义。
Other AbstractBased on our previous work, EaCyp19a1a gene was cloned from the SMART cDNA library of red-spotted grouper oocytes, and the corresponding anti-EaCyp19a1a antiserum was generated for the detection of expression pattern and regulation mechanism in adults. Multiple amino acid sequence alignment of the deduced EaCyp19a1a and other known Cyp19 aromatase proteins revealed high homology among the teleost ovarian isoform, including the common functional domains of aromatases. Moreover, some specific residues were found to be conserved among the Cyp19a1a proteins of teleosts. The detailed localization and dynamic expression change were observed in the gonads of protogynous hermaphrodite red-spotted grouper by utilizing the advantages that gonad development undergoes transition from ovary to intersexual gonad and then to testis. Studies revealed ovary-specific expression pattern of EaCyp19a1a in adults. And EaCyp19a1a was expressed by follicular cells of follicular layer around oocytes. During artificial sex reversal, EaCyp19a1a expression dropped significantly from female to male, and almost no any positive EaCyp19a1a signal was observed in testicular tissues. Another significant progress in this study was to isolate and sequence the promoter region of EaCyp19a1a. Sequence analysis showed twenty-seven potential binding sites were on the promoter for some transcriptional factors, such as SF-1, CREB, SOX5, and so on. luciferase assays in vitro indicated that the CREB regulation region from -1010 to -898 might be a major cis-acting element to EaCyp19a1a promoter. Moreover, specific green fluorescence was observed mainly in primordial gonadal cells of the zebrafish embryos after microinjection in vivo, which indicated that some cis-acting elements were binding to EaCyp19a1a promoter and had the ability to regulate its expression specifically in gonad. Moreover, we found a high degree of conserved region about 300 bp existed in the proximal Cyp19a1a promoters of teleost fishes. And the motifs of TATA box, SF-1, SOX5, and CREB were most notably well-conserved in this region. In view of the Cyp19 studies in human, we suggested the conserved promoter region among teleosts might be important for the ovary-specific expression of Cyp19a1a.
Document Type学位论文
Recommended Citation
GB/T 7714
黄伟. 赤点石斑鱼卵型芳香化酶基因的表达特征及其启动子功能分析[D]. 水生生物研究所. 中国科学院水生生物研究所,2009.
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