利用模式动物斑马鱼研究基因FoxP4在心脏发育中的作用
发布时间：2018-08-03 13:27
【摘要】：本文利用斑马鱼模型研究了FoxP4基因在心脏发育中的调控功能。通过酵母双杂交技术筛选获得了与FoxP4相互作用蛋白CatD、Tcap和FBXL5,进一步研究了FoxP4及其相互作用蛋白CatD、Tcap和FBXL5在心脏发育中的调控功能。 人类FoxP4基因位于第6号染色体上,生物信息学分析表明该基因cDNA长5965bp,共有17个外显子,在基因组上的跨度达54kb,编码一个长为680个氨基酸的蛋白质,它包含一个N-端的C2H2型锌指结构和一个C-端的Fork Head结构域,具有结合DNA的功能,属于具有侧翼螺旋结构的Foxp亚族的转录因子,与亚族中的FoxP1、FoxP2有很高的同源度。RT-PCR和Northern Blot实验发现人类FoxP4基因在人类胚胎发育过程各组织,如心脏、大脑、肺、肝脏和肠等中广泛表达。胚胎原位杂交实验也表明,斑马鱼FoxP4基因在胚胎发育早期持续表达。这说明FoxP4基因是早期胚胎发育中各个组织所必需的转录因子。通过酵母双杂交技术,我们筛选和鉴定了FoxP4的三个相互作用蛋白CatD、Tcap和FBXL5,免疫共沉淀和亚细胞共定位都证明FoxP4与这三个蛋白确实存在相互作用。 利用模式动物斑马鱼以及GFP特异标记心脏的转基因斑马鱼nppa: GFP模型,我们对FoxP4及其相互作用的基因CatD、Tcap和FBXL5的功能进行了研究。RNA干扰和Morpholino敲减实验发现,敲减FoxP4、CatD、Tcap的蛋白表达或增强FBXL5的mRNA表达,斑马鱼胚胎的心脏表现出畸形,如心房增大、心律失常、心脏未能环化等症状,这提示FoxP4及其相互作用基因CatD、Tcap和FBXL5,参与早期心脏的形态建成,在心脏发育信号通路中必不可少。RT-PCR实验结果显示,敲减FoxP4及其相互作用基因CatD和Tcap的蛋白表达,会使Nkx2.5调控的心脏信号通路受到阻碍,影响下游基因的表达,如cardiac-actin和Nkx2.5的表达都发生显著改变,从而使心脏的发育受到影响,产生畸形,证明FoxP4及其相互作用基因CarD和Tcap位于Nkx2.5调控的心脏信号通路的下游,受Nkx2.5的调控；FoxP4和CatD可能处于nkx2.5和cardiac-actin之间的位置。另外,RT-PCR、荧光素酶报告系统分析实验以及Western blot实验都表明FBXL5参与Nkx2.5的调控。在细胞中增强FBXL5的表达,会使Nkx2.5的增强子活性增强,而随着FBXL5蛋白表达的增强, Nkx2.5的表达量增多。总之,通过以上研究,我们认为FoxP4及其相互作用基因CatD、Tcap和FBXL5对心脏发育具有调控作用,这种调控作用可能是通过Nkx2.5信号通路而实现的。 nppa:GFP转基因斑马鱼是通过包含日本青溕Tol2转座子和GFP报告蛋白的增强子诱捕载体,将GFP插入到nppa增强子的附近,使得GFP表达受到nppa增强子的调控而特异地只在心脏表达而筛选得到的。利用nppa:GFP转基因斑马鱼我们不仅研究了FoxP4及其相互作用基因的功能,同时我们也从形态学和生理学上分析了nppa:GFP转基因鱼的心脏功能。与野生型斑马鱼比较,没有发现此转基因鱼心律失常,心率和心跳周期没有差异,心室最大舒张容积和最小收缩容积也无差别,心输出量无差异。因此nppa:GFP转基因鱼的生理功能与野生型鱼无差异,可以用于在体内研究分析由nppa调控的心脏发育和心脏疾病的新型模型,也可以用于检测和高通量筛选心脏疾病治疗的药物。
[Abstract]:In this paper, a zebrafish model was used to study the regulatory function of FoxP4 gene in the development of the heart. FoxP4 interacting protein CatD, Tcap and FBXL5 were screened by yeast two hybrid technique. The regulatory functions of FoxP4 and its interacting protein CatD, Tcap and FBXL5 in the development of heart were further studied.
The human FoxP4 gene is located on chromosome sixth. Bioinformatics analysis shows that the gene cDNA is long 5965bp, with a total of 17 exons, and the span of the genome is 54kb, encoding a protein with a length of 680 amino acids, which contains a C2H2 zinc finger structure at the N- end and a Fork Head domain of the C- end, with the function of binding DNA. The transcription factors of the Foxp subgroup with the flanking spiral structure, the high homology.RT-PCR and the Northern Blot in the subgroup of FoxP1, FoxP2, and Northern Blot found that the human FoxP4 gene is widely expressed in the human embryonic development processes, such as the heart, the brain, the lung, the liver and the intestines. The in situ hybridization experiment also showed that the zebrafish FoxP4 gene was also found. The FoxP4 gene is a necessary transcription factor in the early embryonic development. We screened and identified three interacting proteins, CatD, Tcap and FBXL5, and both FoxP4 and the three proteins. Interaction.
Using the model animal zebrafish and the transgenic zebrafish nppa: GFP model with GFP specifically labeled heart, we have studied the functions of FoxP4 and its interacting genes, CatD, Tcap and FBXL5, to study the.RNA interference and Morpholino knockout experiments, and the expression of the protein expression of the knockout FoxP4, CatD, Tcap, or the zebrafish embryo. Cardiac abnormalities, such as atrial enlargement, arrhythmia, and failure of the heart, suggest that FoxP4 and its interacting genes, CatD, Tcap and FBXL5, are involved in early cardiac morphogenesis, and the essential.RT-PCR experimental results in the cardiac signaling pathway show that the protein expression of FoxP4 and its interacting genes, CatD and Tcap, are expressed, The cardiac signaling pathway regulated by Nkx2.5 is hindered, which affects the expression of the downstream genes, such as the expression of cardiac-actin and Nkx2.5, which affects the development of the heart and produces deformities. It proves that the FoxP4 and its interacting genes, CarD and Tcap, are downstream of the cardiac signaling pathway regulated by Nkx2.5 and are regulated by Nkx2.5. FoxP4 and CatD may be in the position between Nkx2.5 and cardiac-actin. In addition, RT-PCR, Luciferase Report System Analysis and Western blot experiments show that FBXL5 is involved in Nkx2.5 regulation. The enhancement of FBXL5 expression in cells will enhance the activity of Nkx2.5 enhancers, and the expression of FBXL5 protein expression increases. In a word, through these studies, we believe that FoxP4 and its interacting genes, CatD, Tcap and FBXL5, have a regulatory role in the development of the heart, which may be achieved through the Nkx2.5 signaling pathway.
The nppa:GFP transgenic zebrafish is an enhancer entrapment vector containing the Tol2 transposon and GFP reporter protein of Japan, inserting GFP into the vicinity of the NPPA enhancer, making the GFP expression screened by the NPPA enhancer and specifically only in the heart expression. Using nppa: GFP transgenic zebrafish we not only studied FoxP4. At the same time, we also analyzed the cardiac function of nppa:GFP transgenic fish in morphology and physiology. Compared with wild zebrafish, no arrhythmia was found, heart rate and heartbeat cycle were not different, the maximum ventricular diastolic volume and minimum systolic volume were not different, and the cardiac output was not poor. Therefore, the physiological function of nppa:GFP transgenic fish is not different from that of wild type fish. It can be used in the study of new models of heart development and heart disease regulated by NPPA in vivo, and can also be used to detect and high throughput screening drugs for heart disease.
【学位授予单位】：湖南师范大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R33
[Abstract]:In this paper, a zebrafish model was used to study the regulatory function of FoxP4 gene in the development of the heart. FoxP4 interacting protein CatD, Tcap and FBXL5 were screened by yeast two hybrid technique. The regulatory functions of FoxP4 and its interacting protein CatD, Tcap and FBXL5 in the development of heart were further studied.
The human FoxP4 gene is located on chromosome sixth. Bioinformatics analysis shows that the gene cDNA is long 5965bp, with a total of 17 exons, and the span of the genome is 54kb, encoding a protein with a length of 680 amino acids, which contains a C2H2 zinc finger structure at the N- end and a Fork Head domain of the C- end, with the function of binding DNA. The transcription factors of the Foxp subgroup with the flanking spiral structure, the high homology.RT-PCR and the Northern Blot in the subgroup of FoxP1, FoxP2, and Northern Blot found that the human FoxP4 gene is widely expressed in the human embryonic development processes, such as the heart, the brain, the lung, the liver and the intestines. The in situ hybridization experiment also showed that the zebrafish FoxP4 gene was also found. The FoxP4 gene is a necessary transcription factor in the early embryonic development. We screened and identified three interacting proteins, CatD, Tcap and FBXL5, and both FoxP4 and the three proteins. Interaction.
Using the model animal zebrafish and the transgenic zebrafish nppa: GFP model with GFP specifically labeled heart, we have studied the functions of FoxP4 and its interacting genes, CatD, Tcap and FBXL5, to study the.RNA interference and Morpholino knockout experiments, and the expression of the protein expression of the knockout FoxP4, CatD, Tcap, or the zebrafish embryo. Cardiac abnormalities, such as atrial enlargement, arrhythmia, and failure of the heart, suggest that FoxP4 and its interacting genes, CatD, Tcap and FBXL5, are involved in early cardiac morphogenesis, and the essential.RT-PCR experimental results in the cardiac signaling pathway show that the protein expression of FoxP4 and its interacting genes, CatD and Tcap, are expressed, The cardiac signaling pathway regulated by Nkx2.5 is hindered, which affects the expression of the downstream genes, such as the expression of cardiac-actin and Nkx2.5, which affects the development of the heart and produces deformities. It proves that the FoxP4 and its interacting genes, CarD and Tcap, are downstream of the cardiac signaling pathway regulated by Nkx2.5 and are regulated by Nkx2.5. FoxP4 and CatD may be in the position between Nkx2.5 and cardiac-actin. In addition, RT-PCR, Luciferase Report System Analysis and Western blot experiments show that FBXL5 is involved in Nkx2.5 regulation. The enhancement of FBXL5 expression in cells will enhance the activity of Nkx2.5 enhancers, and the expression of FBXL5 protein expression increases. In a word, through these studies, we believe that FoxP4 and its interacting genes, CatD, Tcap and FBXL5, have a regulatory role in the development of the heart, which may be achieved through the Nkx2.5 signaling pathway.
The nppa:GFP transgenic zebrafish is an enhancer entrapment vector containing the Tol2 transposon and GFP reporter protein of Japan, inserting GFP into the vicinity of the NPPA enhancer, making the GFP expression screened by the NPPA enhancer and specifically only in the heart expression. Using nppa: GFP transgenic zebrafish we not only studied FoxP4. At the same time, we also analyzed the cardiac function of nppa:GFP transgenic fish in morphology and physiology. Compared with wild zebrafish, no arrhythmia was found, heart rate and heartbeat cycle were not different, the maximum ventricular diastolic volume and minimum systolic volume were not different, and the cardiac output was not poor. Therefore, the physiological function of nppa:GFP transgenic fish is not different from that of wild type fish. It can be used in the study of new models of heart development and heart disease regulated by NPPA in vivo, and can also be used to detect and high throughput screening drugs for heart disease.
【学位授予单位】：湖南师范大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R33
【共引文献】
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