利用模式動(dòng)物斑馬魚研究基因FoxP4在心臟發(fā)育中的作用
發(fā)布時(shí)間：2018-08-03 13:27
【摘要】：本文利用斑馬魚模型研究了FoxP4基因在心臟發(fā)育中的調(diào)控功能。通過酵母雙雜交技術(shù)篩選獲得了與FoxP4相互作用蛋白CatD、Tcap和FBXL5,進(jìn)一步研究了FoxP4及其相互作用蛋白CatD、Tcap和FBXL5在心臟發(fā)育中的調(diào)控功能。 人類FoxP4基因位于第6號(hào)染色體上,生物信息學(xué)分析表明該基因cDNA長5965bp,共有17個(gè)外顯子,在基因組上的跨度達(dá)54kb,編碼一個(gè)長為680個(gè)氨基酸的蛋白質(zhì),它包含一個(gè)N-端的C2H2型鋅指結(jié)構(gòu)和一個(gè)C-端的Fork Head結(jié)構(gòu)域,具有結(jié)合DNA的功能,屬于具有側(cè)翼螺旋結(jié)構(gòu)的Foxp亞族的轉(zhuǎn)錄因子,與亞族中的FoxP1、FoxP2有很高的同源度。RT-PCR和Northern Blot實(shí)驗(yàn)發(fā)現(xiàn)人類FoxP4基因在人類胚胎發(fā)育過程各組織,如心臟、大腦、肺、肝臟和腸等中廣泛表達(dá)。胚胎原位雜交實(shí)驗(yàn)也表明,斑馬魚FoxP4基因在胚胎發(fā)育早期持續(xù)表達(dá)。這說明FoxP4基因是早期胚胎發(fā)育中各個(gè)組織所必需的轉(zhuǎn)錄因子。通過酵母雙雜交技術(shù),我們篩選和鑒定了FoxP4的三個(gè)相互作用蛋白CatD、Tcap和FBXL5,免疫共沉淀和亞細(xì)胞共定位都證明FoxP4與這三個(gè)蛋白確實(shí)存在相互作用。 利用模式動(dòng)物斑馬魚以及GFP特異標(biāo)記心臟的轉(zhuǎn)基因斑馬魚nppa: GFP模型,我們對(duì)FoxP4及其相互作用的基因CatD、Tcap和FBXL5的功能進(jìn)行了研究。RNA干擾和Morpholino敲減實(shí)驗(yàn)發(fā)現(xiàn),敲減FoxP4、CatD、Tcap的蛋白表達(dá)或增強(qiáng)FBXL5的mRNA表達(dá),斑馬魚胚胎的心臟表現(xiàn)出畸形,如心房增大、心律失常、心臟未能環(huán)化等癥狀,這提示FoxP4及其相互作用基因CatD、Tcap和FBXL5,參與早期心臟的形態(tài)建成,在心臟發(fā)育信號(hào)通路中必不可少。RT-PCR實(shí)驗(yàn)結(jié)果顯示,敲減FoxP4及其相互作用基因CatD和Tcap的蛋白表達(dá),會(huì)使Nkx2.5調(diào)控的心臟信號(hào)通路受到阻礙,影響下游基因的表達(dá),如cardiac-actin和Nkx2.5的表達(dá)都發(fā)生顯著改變,從而使心臟的發(fā)育受到影響,產(chǎn)生畸形,證明FoxP4及其相互作用基因CarD和Tcap位于Nkx2.5調(diào)控的心臟信號(hào)通路的下游,受Nkx2.5的調(diào)控；FoxP4和CatD可能處于nkx2.5和cardiac-actin之間的位置。另外,RT-PCR、熒光素酶報(bào)告系統(tǒng)分析實(shí)驗(yàn)以及Western blot實(shí)驗(yàn)都表明FBXL5參與Nkx2.5的調(diào)控。在細(xì)胞中增強(qiáng)FBXL5的表達(dá),會(huì)使Nkx2.5的增強(qiáng)子活性增強(qiáng),而隨著FBXL5蛋白表達(dá)的增強(qiáng), Nkx2.5的表達(dá)量增多�？傊�,通過以上研究,我們認(rèn)為FoxP4及其相互作用基因CatD、Tcap和FBXL5對(duì)心臟發(fā)育具有調(diào)控作用,這種調(diào)控作用可能是通過Nkx2.5信號(hào)通路而實(shí)現(xiàn)的。 nppa:GFP轉(zhuǎn)基因斑馬魚是通過包含日本青溕Tol2轉(zhuǎn)座子和GFP報(bào)告蛋白的增強(qiáng)子誘捕載體,將GFP插入到nppa增強(qiáng)子的附近,使得GFP表達(dá)受到nppa增強(qiáng)子的調(diào)控而特異地只在心臟表達(dá)而篩選得到的。利用nppa:GFP轉(zhuǎn)基因斑馬魚我們不僅研究了FoxP4及其相互作用基因的功能,同時(shí)我們也從形態(tài)學(xué)和生理學(xué)上分析了nppa:GFP轉(zhuǎn)基因魚的心臟功能。與野生型斑馬魚比較,沒有發(fā)現(xiàn)此轉(zhuǎn)基因魚心律失常,心率和心跳周期沒有差異,心室最大舒張容積和最小收縮容積也無差別,心輸出量無差異。因此nppa:GFP轉(zhuǎn)基因魚的生理功能與野生型魚無差異,可以用于在體內(nèi)研究分析由nppa調(diào)控的心臟發(fā)育和心臟疾病的新型模型,也可以用于檢測和高通量篩選心臟疾病治療的藥物。
[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.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：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.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R33
【共引文獻(xiàn)】
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1 袁婺洲;唐e，
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