赤点石斑鱼促甲状腺激素TSHβ启动子的克隆及其在斑马鱼胚胎原始性腺和垂体中定位表达  

CLONING OF RED-SPOTTED GROUPER TSHβ PROMOTER AND ITS LOCALIZATION EXPRESSION IN PRIMITIVE GONAD AND PITUITARY OF ZEBRAFISH EMBRYOS

作　　者：李辰昱[1] 汪洋[1] 周莉[1] 桂建芳[1] 

机构地区：[1]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉430072

出　　处：《水生生物学报》2010年第4期822-827,共6页Acta Hydrobiologica Sinica

基　　金：国家973计划项目(NO.2004CB117401);国家自然科学基金(NO.U0631007)资助

摘　　要：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。斜带石斑鱼(Epinephelus coioides)促甲状腺激素TSHβ除在垂体中表达外还在性腺中表达,进一步发现TSHβ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,TSHβ转录水平升高。为研究石斑鱼TSHβ特殊表达方式的调控机制,采用基因组步移技术扩增获得赤点石斑鱼TSHβ5′端序列共5112bp。将1864bp核心片段顺向克隆到pBK-TOL2载体中,pTSHβ-TOL2重组质粒与转座酶mRNA共注射斑马鱼一细胞期受精卵。Western blot结果显示,在受精后10h的尾芽胚中可检测到GFP的表达,胚体晚期GFP表达量最高。荧光显微镜追踪观察发现,受精后12—30hpf(Hours post fertilization)观察到GFP在原始生殖细胞(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。由此可见,赤点石斑鱼TSHβ5′端1864bp的序列具有特异定位的启动子活性。Thyroid-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 were considerably less in lower vertebrates.Our lab had cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone β-subunit（TSHβ） from orange-spotted grouper（Epinephelus coioides）,and first reported TSHβ expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary.Further analysis found that TSHβ also expressed in the gonads of the red-spotted grouper（Epinephelus akaara）.To elucidate the mechanisms regulating TSHβ expression,we isolated a 5.1kb fragment of the 5＇-flanking region of TSHβ promoter from red-spotted grouper by Genome walking.1.8 kb of this upstream region was used to produce GFP reporter construct（pBK-TOL2） for analysis of tissue-specific expression in zebrafish embryos.During 12—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.

关 键 词：石斑鱼 TSHβ 启动子 GFP PGC 

分 类 号：S941[农业科学—水产养殖]