留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码
李石竹, 刘威, 李志, 周莉, 卢建国, 桂建芳. 银鲫Greb1的克隆、表达及功能研究[J]. 水生生物学报, 2019, 43(5): 953-961. DOI: 10.7541/2019.113
引用本文: 李石竹, 刘威, 李志, 周莉, 卢建国, 桂建芳. 银鲫Greb1的克隆、表达及功能研究[J]. 水生生物学报, 2019, 43(5): 953-961. DOI: 10.7541/2019.113
LI Shi-Zhu, LIU Wei, LI Zhi, ZHOU Li, LU Jian-Guo, GUI Jian-Fang. MOLECULAR CLONING, EXPRESSION AND FUNCTION ANALYSIS OF GREB1 IN GIBEL CARP, CARASSIUS GIBELIO[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(5): 953-961. DOI: 10.7541/2019.113
Citation: LI Shi-Zhu, LIU Wei, LI Zhi, ZHOU Li, LU Jian-Guo, GUI Jian-Fang. MOLECULAR CLONING, EXPRESSION AND FUNCTION ANALYSIS OF GREB1 IN GIBEL CARP, CARASSIUS GIBELIO[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(5): 953-961. DOI: 10.7541/2019.113

银鲫Greb1的克隆、表达及功能研究

MOLECULAR CLONING, EXPRESSION AND FUNCTION ANALYSIS OF GREB1 IN GIBEL CARP, CARASSIUS GIBELIO

  • 摘要: 为研究银鲫(Carassius gibelio)雌激素应答基因(Growth regulation by estrogen in breast cancer cell 1, Greb1)的表达特征和功能, 采用RACE方法克隆了银鲫Greb1的全长cDNA (CgGreb1)。CgGreb1的cDNA全长为954 bp, 其中包含一个765 bp的开放阅读框, 编码255个氨基酸。大多数脊椎动物有多个Greb1的变体, 长度为386—1988个氨基酸, 其中CgGreb1是最短的一个。序列比对结果表明脊椎动物Greb1蛋白的N端区域极为保守, 且CgGreb1位于Greb1蛋白的N端区域, 该区域与其他脊椎动物Greb1的同源性超过60%。qRT-PCR结果显示, 在成体组织中, CgGreb1主要在垂体、脑、性腺和肝脏中表达, 其中在垂体中CgGreb1的表达最高; 在注射促黄体生成素释放激素类似物和人绒毛膜促性腺激素后的雌鱼垂体中, CgGreb1的表达逐渐上升随后降低, 并在产卵时维持较高的表达; 在胚胎发育过程中, CgGreb1从50%外包开始表达, 其表达量随胚胎发育而升高, 在受精后24h达到峰值, 随后表达量下降, 在受精后48h不表达。原位杂交结果表明, 在原肠期, CgGreb1信号位于胚层的边缘; 在体节期, CgGreb1信号逐渐增强并出现在神经系统; 在受精后24h, CgGreb1信号在脑和脊髓等中枢神经系统增强; 从受精后30h开始, CgGreb1信号减弱; 在受精后48h, 检测不到CgGreb1信号。在注射CgGreb1 MO的银鲫胚胎中, tshβprlgthα分别标记的3种垂体细胞减少, 证明CgGreb1参与早期银鲫垂体细胞的发育。研究结果为进一步揭示银鲫垂体发育和生长调控机制奠定了基础。

     

    Abstract: Greb1 (Growth regulation by estrogen in breast cancer cell 1), an estrogen stimulated gene, plays critical roles in estrogen response. Previous study indicated that Greb1 was highly expressed in gibel carp (Carassius gibelio) pituitary. The current study cloned and characterized the expression of Carassius gibelio Greb1 (CgGreb1), and investi-gated its function in early embryonic development. The full length cDNA of CgGreb1 was 954 bp, encoding a deduced protein of 255 amino acids. Based on the NCBI database, most vertebrates contained at least one Greb1 isoforms, whose amino acid length ranged from 386—1988 and contained conserved N-terminus. Among those Greb1 homologues, CgGreb1 was the shortest one, locating in the N-terminus of other Greb1 homologues and sharing high identi-ties over 60%. CgGreb1 was dominantly expressed in pituitary, brains, liver and gonads with the highest level in pitui-tary. The expression of CgGreb1 was gradually increased by inducing spawning gibel carp females, peaked at 6 hours post induction, and then decreased and maintained at a relatively high level. During embryogenesis, the expression of CgGreb1 started from 50% epiboly, increased as embryogenesis progression, peaked at 24 hours post fertilization (hpf), and then gradually decreased and was undetectable at 48 hpf. WISH analysis showed that CgGreb1 signal was specifi-cally located in the embryonic margin during gastrulation, gradually distributed in the central neural system during somitogenesis, and was strongly expressed in many brain regions and notochord, but was gradually weakened from 30 hpf and was disappeared at 48 hpf. Knockdown of CgGreb1 specifically decreased the expression of tshβ, prl and gthα but not pomca in producing cells, suggesting that CgGreb1 played an important role in early pituitary development in gibel carp. Our present study provides insights into the molecular mechanism underlying pituitary development and growth regulation in gibel carp.

     

/

返回文章
返回