草鱼激活素Ⅰ型和Ⅱ型受体的cDNA克隆、结构分析及其在垂体细胞中的表达调控的研究

【作者】 宋春雷；

【导师】 周红；

【作者基本信息】 电子科技大学 ， 生物物理学， 2009， 硕士

【摘要】 激活素因其参与生殖功能调控而受到广泛重视,尤其是激活素在卵巢和垂体中发挥作用的生理意义近年来得到了阐明。目前对激活素活性调节的研究主要集中在与其两个调节因子(Follistatin和Inhibin)的相互作用,而针对其细胞信号通路水平(如受体表达)的调控还有待研究。在目前的研究中,编码草鱼激活素IIB型受体(ActRIIB)和激活素IB型受体(ActRIB)的两条cDNA序列被克隆,预测的ActRIIB蛋白含510个氨基酸残基,与其它脊椎动物的ActRIIB有79-90%的同源性;而预测的ActRIB蛋白含505个氨基酸残基,与人、大鼠、小鼠、爪蟾和斑马鱼中的ActRIB有80-96%的相似度。系统进化分析显示两种受体在脊椎动物进化过程中高度保守。序列比对分析显示两个受体都包含有激活素结合所必需的保守氨基酸残基,且都包含特征性的结构域:一个胞外配体结合结构域、一个跨膜结构域和一个胞内丝氨酸/苏氨酸激酶结构域。人和草鱼之间受体功能结构域的高度保守性,为我们在实验中采用人激活素A研究草鱼ActRIIB和ActRIB的表达水平提供了支持。Real-time定量PCR分析显示ActRIIB和ActRIB的转录产物广泛分布于包括垂体在内的所有组织中,尤其是在非性腺组织中的高表达可能与作为研究模型的草鱼出于非性成熟期有关。进一步使用原代细胞静态培养的方法,在垂体水平上检测外源性激活素对其自身受体(ActRIIB和ActRIB)表达的影响,发现激活素可以明显以时间剂量依赖型的方式刺激草鱼ActRIB mRNA的表达,但是不改变ActRIIB的表达水平。该发现表明激活素受体的表达水平可能是草鱼垂体内激活素功能的一个调节位点。同时,采用点突变的方法分别构建了激酶活性缺陷型和结构激活型ActRIB突变表达载体以及激活素结合活性缺陷型ActRIIB突变表达载体,为进一步研究这两种受体的功能打下了基础。更多还原

【Abstract】 Since the initial discovery activin, cumulative evidence further has established that activin and its two functional modulators, the activin binding protein follistatin and the activin antagonist inhibin, regulated reproductive function by exerting effects at all levels of the reproductive axis, in particular at the ovary and pituitary. However, the intracellular mechanisms for activin actions, e.g. receptor expression levels, have not been examined. In the present study,two cDNAs encoding the activin type IIB receptor (ActRIIB) and activin type IB receptor (ActRIB) were cloned and characterized from grass carp. The deduced ActRIIB protein of 510 amino acids shared 79-90% identity with those in other vertebrates, while the predicted ActRIB protein of 505 amino acids exhibited high sequence identity (80-96%) to its counterparts in human, rat, mouse, frog and zebrafish. Phylogenetic analysis revealed that two receptors were highly conserved during vertebrate evolution. Furthermore, comparative analysis showed that each of these two receptors contained the conserved amino acid residues required for activin binding, and comprised the characteristic regions of an extracellular ligand binding domain, a single transmembrane region, and an intracellular serine/threonine kinase domain. The high degree of conservation of receptor functional domains between human and grass carp supports the idea that human activin-A used in the present study could interact with grass carp ActRIIB and ActRIB. Real-time PCR analysis revealed that both ActRIIB and ActRIB transcripts were ubiquitously expressed in all of the tissues examined, including pituitary. Using a static incubation approach, the direct actions of exogenous activin on its own receptors, ActRIIB and ActRIB, mRNA expression were examined at the pituitary level. Activin significantly stimulated mRNA expression of grass carp ActRIB in a time- and dose-dependent manner, but didn’t alter the expression level of ActRIIB. These findings support the notion that activin receptors may serve as a local regulatory point involving pituitary function of activin in grass carp. In addition, expression plasmids for kinase deficient form and constitutively active form of ActRIB, and the ligand-binding inefficient form of ActRIIB were constructed using site-directed mutagenesis, respectively, providing the molecular tool for further study on the functional roles of these two receptors.更多还原