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Alternative TitleCloning and activity analysis of TSHb promoter in red-spotted grouper
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石斑鱼 Tshb 启动子 Gfp Pgc
Abstract促甲状腺激素(thyroid-stimulating hormone (TSH))具有调节生长,发育,代谢和生殖的功能, 但是在低等脊椎动物中的功能研究较少。本试验室汪洋等在研究斜带石斑鱼(Epinephelus coioides)TSHb时,首次发现其TSHb除在垂体中表达外还在性腺中表达,进一步发现TSHb也在赤点石斑鱼(Epinephelus akaara)性腺中表达,且在人工诱导的赤点石斑鱼中,随着雄性化的转变,TSHb转录水平升高。为研究石斑鱼TSHb特殊表达的调控机制,采用基因组步移技术(Genome walker),通过5轮扩增获得赤点石斑鱼TSHb启动子片段,共5112bp。将起始密码子上游1864bp的启动子片段顺向克隆到pBK-TOL2载体中, pTSHb-TOL2重组质粒与转座酶mRNA共注射斑马鱼一细胞期受精卵,荧光显微镜观察发现,TSHb启动子驱动GFP在原始生殖细胞(PGC)和垂体中表达。受精后12hpf-30hpf观察到GFP在PGC区域表达,36hpf-7dpf观察到GFP除在PGC区域表达外还在垂体中表达。Western blot结果显示,尾芽期可检测到GFP的表达,胚体晚期GFP表达量最高。将一系列启动子缺失片断顺向克隆到pGL-3BGFP载体中,通过显微注射斑马鱼一细胞期受精卵对TSHb基因的启动子进行缺失分析表明,起始密码子ATG上游(-613~-1bp)片断为启动子具有活性所必需。与PGC的标记基因VASA共定位的结果显示,TSHb启动子驱动GFP表达于PGC的周围。
Other AbstractThyroid-stimulating hormone (TSH) regulates the growth and function of thyroid for formation of thyroid hormones, which are involved in regulation of growth, development, metabolism and reproduction. TSHβ and their thyroidal receptors have been investigated extensively in mammals, but comparative studies in lower vertebrates have been considerably less. Our lab had first reported thyroid stimulating hormone b-subunit (TSHb) expressed in the gonads and pituitary of the orange-spotted grouper (Epinephelus coioides), Further analysis found TSHb expressed in the gonads of the red-spotted grouper (Epinephelus akaara).To elucidate the mechanisms regulating TSHb expression, we isolated a 5.1-kb fragment of the red-spotted grouper 5’-flanking region by Genome walking .1.8kb of this upstream region was used to produce GFP reporter construct (pBK-TOL2) for analysis of tissue-specific expression in zebrafish embryos. During 12hpf-30hpf stage, GFP expressed in the PGC; 36hpf-7dpf stage, GFP expressed not only in the PGC but also in pituitary. Western blot analysis showed that GFP protein was detected early in the 10hpf stage embryos. Five sequentially deleted fragments of this upstream region were used to produce GFP reporter constructs (pGL-3BGFP) for analysis of tissue-specific expression in zebrafish embryos. The results also show that a 613-bp upstream fragment is sufficient to direct strong expression of the GFP in gonads but also in pituitary. The Co-localization of TSHb promoter with that of VASA, shown TSHb promoter directed GFP express close to PGC.
Document Type学位论文
Recommended Citation
GB/T 7714
李辰昱. 赤点石斑鱼促甲状腺激素基因TSHb启动子的克隆及其活性分析[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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