导　　师： 吴秀山;邓云

学科专业： 071007

授予学位： 硕士

作　　者： ;

机构地区： 湖南师范大学

摘　　要： 心脏是脊椎动物最早发育并行驶功能的器官之一,其发育过程受到多种基因的表达调控。这些基因的表达异常会导致不同类型的心脏缺陷。在我国约有1000万先天性心脏病患者,这类疾病已上升为人类的头号杀手,因此,研究心脏发育的调控基因,有助于更好地了解心脏病的发病机制以及对该类疾病的防治。斑马鱼作为一种理想的模式动物,具有个体小、易于饲养、发育快、繁殖能力强、体外受精、胚胎体外发育且透明等优点,其基因组与人类的相似性达到了87/%,因此使用斑马鱼作为脊椎动物发育生物学模型研究心脏发育的基因调控机制具有极大的优势。 本室的前期研究显示,斑马鱼基因HPRG1可能参与心脏早期的形态建成,与Smad蛋白相互作用,通过BMP信号途径在心脏发育中发挥重要的调控作用。本论文以斑马鱼为模型继续研究HPRG1基因在脊椎动物心脏发育中的功能。 本论文首先通过整体胚胎原位杂交研究HPRG1基因在斑马鱼胚胎发育中的表达模式。实验结果表明,HPRG1基因在胚胎发育的早期开始表达,在发育晚期定位于心脏组织特异性表达,这与本室前期的胚胎免疫荧光实验结果相符合。其次,本文利用To12转座子系统构建了一系列与斑马鱼心脏候选基因HPRG1相关的转基因斑马鱼品系,其中已成功建立的可稳定遗传的品系包括：Tg:pTol2/(cmlc2:HPRG1-EGFP/)、Tg: pTol2/(HPRG1/(3kb/):EGFP/), Tg:pTol2/(HPRG1/(2kb/): EGFP/).pTol2/(HPRG1/(1kb/):EGFP/)。通过对pTol2/(cmlc2:HPRG1-EGFP/)过表达品系进行表型分析,发现HPRG1基因在心脏过表达导致围心腔变大、心脏不能正常环化及心室缩小、心房膨大等缺陷表型。利用斑马鱼M-mode生理学研究技术,进一步证明HPRG1基因对心脏生理学功能起着重要的调控作用。对pTol2/(HPRG1/(3kb/): EGFP/)品系的荧光表达模式分析发现,3kb的启动子限制HPRG1基因在心脏组织特异性表达,其表达模式与之前的原位杂交表达模式相符合。另外,本文还利用To12转座子系统建立了pTol2/(cmlc2: CRE-IRES-EGFP/)转基因斑马鱼品系。分析表明,该品系在心脏特异性表达CRE重组酶,对心脏的正常发育没有明显影响。该品系的成功建立为利用条件基因打靶技术研究斑马鱼候选基因在心脏发育与相关疾病中的功能奠定了基础。 本论文利用近几年兴起的CRISPR//Cas9打靶技术建立斑马鱼HPRG1基因敲除系。首先经网站分析筛选出HPRG1基因最合适的打靶位点,构建出HPRG1基因的左右半位点CRSPR表达载体,转录成RNA并与Cas9核酸内切酶的mRNA相混合,显微注射至斑马鱼单细胞期胚胎。以提取的24hpf胚胎基因组为模板进行PCR扩增和酶切检测,证明该表达载体能有效地诱发基因突变。斑马鱼HPRG1基因的稳定敲除模型正在构建中。 Heart is one of the earliest developmental and functional organs of vertebrate, and its development process is regulated by a lot of genes. The abnormal expression of these genes may result in different cardiac defects. In our country, there are about10million patients suffer from congenital heart disease and this kind of disease is known as the number one killer of human. Therefore, researches on the regulatory genes of cardiac development help to better understand the pathogenesis of heart disease and is of great significance for the prevention and treatment of the disease. Zebraflsh, as a kind of ideal animal models, has advantages such as individual small, easy to breed, rapid development, strong reproductive capacity, in vitro fertilization, embryo development in vitro and transparent, and its genome similarity compared with human is as much as87/%, so using zebrafish as a vertebrate developmental biology model to study the mechanism of gene regulation of cardiac development has a big superiority. Our previous studies showed that the zebrafish gene HPRG1may be involved in the early morphogenesis of the heart and affected the BMP signaling pathways that plays an important role in heart development by interacting with Smad proteins. In this paper, zebrafish was used as a model for further study of the functions of HPRG1in vertebrate heart development. This paper first studied in HPRG1gene expression pattern in zebrafish embryonic development through the use of whole embryo in situ hybridization. The experimental results showed that the HPRG1gene expressed at the early stages of embryonic development, and specifically expressed in the heart tissues at later stages. These results are coincident with the previous results of embryonic immunofluorescence experiment. Moreover, this paper uses To12transposon system to generate a series of transgenic zebrafish lines related to zebrafish heart candidate gene HPRG1, including:Tg: pTol2/(cmlc2: HPRGl-EGFP/), Tg: PTol2/(HPRG1/(3kb/):EGFP/), Tg: pTol2/(HPRG1/(2kb/):EGFP/), PTol2/(HPRG1/(1kb/):EGFP/). The results of phenotypic analysis of pTol2/(cmlc2:HPRG1-EGFP/) line shows that overexpression of HPRG1in heart leads to many cardiac defects, including larger pericardial cavity, abnormal cardiac cyclization, smaller ventricle and larger atrium.The subsequent M-mode physiological analysis further proved that the HPRG1gene played an important role in normal development of the heart. The results of phenotypic analysis of the pTol2/(HPRG1/(3kb/): EGFP/) line showed that3kb promoter restricted HPRGl gene expressed in the heart tissues specifically, and its expression pattern is coincident with the previous result of in situ hybridization. In addition, this paper also generated a pTol2/(cmlc2: CRE-IRES-EGFP/) line, which specifically expressed Cre recombinant enzyme in heart and has no significant effect on the normal development of the hear.Thus, the successful generation of this transgenic zebrafish line laided foundation for the study of the function of heart development candidate genes in heart development and related heart diseases. In this paper, CRISPR//Cas9targeting technology was used to knockout zebrafish gene HPRG1. Through the website analysis, we identified the most appropriate target site of the HPRG1gene and constructed the left and right half sites CRISPR expression plasmids, which was then transcribed into RNA and microinjected into zebrafish one cell phase embryos mixed with Cas9endonuclease mRNA. With using the extracted genome of24hpf embryos as template for PCR amplification and subsequent enzyme digestion,we confirmed that the expression vectors can induce gene mutation effectively.The generation of stable knockout model of zebrafish gene HPRG1is underway.

关 键 词： 斑马鱼 转基因 心脏发育

分 类 号： [S917.4]

领　　域： [农业科学]