同源框基因HOXA11启动子重组质粒的构建与表达

发布时间：2018-02-09 06:23

本文关键词： HOXA11基因启动子 T载体 pEGFP-1 Ishikawa细胞　出处：《大连医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 同源框基因(homeobox gene)是一类调控胚胎发育和细胞分化的转录调节基因,这类基因中都存在一个由183个碱基对(bp)组成的、高度保守的同源框(homeobox)序列。HOX基因是同源框基因中的一大类,其表达的蛋白质是一类转录调节因子,都有一个由相应同源框序列编码的约61个氨基酸组成的同源域(homeodomain)结构,这一结构通过与顺式调控元件相结合,实现HOX基因对靶基因的调控。HOXA11属HOX基因家族中的一员,在胚胎尾部体节中表达,主要参与子宫形态的发生和发育;HOXA11基因的产物作为转录因子参与调节子宫内膜形态发育和分化成熟。卵巢激素与HOX基因表达的关系成为近年来生殖领域的研究热点。目前认为HOX基因是性激素对子宫调节链中的重要介导基因,雌、孕激素对内膜的多种调节作用主要通过HOX基因介导完成的。本课题组以往的研究发现,HOXA11在子宫内膜的表达受雌、孕激素调节,在分泌中期HOXA11表达变化最明显;并且首次发现,分泌中期内膜腺上皮HOXA11的表达明显减弱甚至消失、孕激素对内膜腺上皮HOXA11基因的表达具有负调控作用。这种负调控作用与分泌中期内膜的分化、成熟以及子宫接受态(receptive state)的形成密切相关。但是,有关孕激素对内膜腺上皮HOXA11基因的负调控作用机制,目前尚不清楚;HOXA11基因的启动子区是否存在着孕激素反应元件尚未见报道。因此,本研究的目的是:通过构建HOXA11 pEGFP-启动子重组质粒,确定HOXA11基因启动子的位置;证明HOXA11启动子区是否存在孕激素反应元件,为进一步研究孕激素对内膜腺上皮负调控机制奠定实验基础。主要方法: 从基因库中查找人HOXA11基因序列,分别在正义链和反义链上预测两段启动子区域(分别命名为启动子1,启动子2),设计PCR引物;从人蜕膜组织中提取基因组DNA,用PCR方法得到克隆目的基因片段;
[Abstract]:Homeobox gene (homeobox gene) is a kind of transcriptional regulation of embryonic development and cell differentiation regulation genes, these genes are one of the 183 base pairs (BP) composed of highly conserved homeobox.HOX gene sequence (homeobox) is a large class of homeobox genes, the expression of protein is a transcription factor, a homeodomain by a corresponding homeobox encoding a sequence of about 61 amino acids (homeodomain) structure, this structure is combined with cis regulatory elements, a member of the regulation of HOX gene on.HOXA11 gene belongs to HOX gene family, expressed in embryonic tail somite, mainly involved in the occurrence and development of uterine morphology; HOXA11 gene product as a transcription factor involved in the regulation of endometrial morphological development and differentiation. The relationship between ovarian hormones and HOX gene expression has become the field of reproductive research in recent years Hotspot. Now that the HOX gene is an important regulation of sex hormones on uterine lies in the chain of gene, estrogen, progesterone on endometrial variety regulation is mainly mediated by HOX gene complete. Our group's previous study found that HOXA11 expression in endometrial estrogen and progesterone regulate in mid secretory expression of HOXA11 the most obvious change; and for the first time found that the expression of the mid secretory endometrial epithelial cells of HOXA11 decreased and even disappeared with the negative regulation of progesterone on the expression of endometrial epithelial cells. HOXA11 gene differentiation and secretion of the negative regulation of the medium term membrane, maturation and uterine receptivity (receptive state) are closely related. However, negative the regulation mechanism of progesterone on endometrial epithelial HOXA11 gene, it is unclear; the promoter region of HOXA11 gene and the existence of progesterone response element has not been reported for. The purpose of this study is to identify the location of the promoter of HOXA11 gene by constructing the recombinant plasmid of HOXA11 pEGFP- promoter, and to prove whether there is progesterone response element in HOXA11 promoter region, so as to lay an experimental foundation for further study of the negative regulation mechanism of progesterone on the endometrial glandular epithelium.
The main methods:
The sequence of human HOXA11 gene was searched from the gene pool, and the two promoter regions were predicted on the chain of justice and antisense chain, respectively, named as promoter 1, promoter 2. PCR primers were designed, genomic DNA was extracted from human decidual tissue, and the target gene fragment was obtained by PCR.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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