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標題: | 針對udu和udub基因的生化區域與發育功能之分析 The biochemical domains analysis and developmental functions of udu and udub genes |
作者: | I-Chieh Chiang 姜以倢 |
指導教授: | 江運金(Yun-Jin Jiang) |
關鍵字: | 斑馬魚,血球發育,血管發育,血管新生,細胞凋亡, zebrafish,udu,udub,hematopoiesis,angiogenesis,apoptosis, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 斑馬魚Udu 為人類GON4L的同源蛋白,編碼為一種核因子,其對於血管新生、造血作用以及細胞週期扮演重要的角色。在Udu蛋白中,目前被發現具有三種保留性區域:YY1BD (YY1-binding domain)、PAH (two paired amphipathic alpha –helix-like)以及SANT ( Swi3, Ada2, N-CoR, and TFIIIB-like)。Udu/GON4L的功能在其他物種中參與了不同的表型,而我們認為很可能是透過缺乏不同的保留性區域所導致。為了研究Udu中各個保留性區域的功能,我們使用了全長的Udu、N端Udu (具有兩個YY1BD)以及C端Udu (擁有PAH和SANT區域)進行mRNA的過度表達實驗。實驗的結果顯示,透過全長udu mRNA的注射可以拯救udutu24突變體所具有的造血作用和細胞凋亡缺陷,並且可以透過注射N端udu mRNA挽救血管生成的缺陷。為了進一步研究保留性區域的功能以及Udu與Udub之間的關係,我們利用CRISPR 系統在斑馬魚中進行基因敲除 (KO) 並在udu與udub中產生具有不同保留性區域的無義突變 (nonsense mutation),在udunn11突變體中(具有兩個YY1BD),與其他udu突變體一樣具有造血功能和血管生成缺陷並在早期發育階段中死亡。另一方面,在胚胎發育的過程中,udub基因敲除品系與其同齡的野生種之間並無顯著差異,但在3個月大的斑馬魚中我們發現udub突變體大量的死亡,並且具有較短的體長。於是我們對udub突變種進行解剖以及透過H&E、Sirius red以及PCNA染色,我們發現成魚的肝臟看似異常並且具有較低的細胞增生訊號。最後,我們發現udubnn32突變體中的udu RNA表現量下降,這顯示Udub有可能影響udu的mRNA表達。
總結來說,udutu24,udusq3和udunn11在胚胎發育過程中具有相似的缺陷,而udub的突變體主要影響成魚的發育以及死亡,也顯示著udu和udub分别在胚胎階段以及成魚階段分別扮演了很重要的角色。 Udu, a homolog of human GON4L, is a protein encodes a novel nuclear factor that has critical roles for angiogenesis, hematopoiesis, cell cycle, DNA replication, etc. The conserved domains in Udu include two YY1BD (YY1-binding domain), two PAH repeats (two paired amphipathic alpha –helix-like) and a SANT (Swi3, Ada2, N-CoR, and TFIIIB-like) domain. The function of Udu/GON4L participates in different phenotypes in other species, and we think it is likely to be caused by the lack of different conserved domains. To investigate the functions of different domains in Udu, N-terminus of Udu containing two YY1BD, and C-terminus of Udu containing PAH and SANT domains were used. While overexpressing the udu mRNA, hematopoiesis and apoptosis defects in udutu24 mutants were rescued with full-length udu and the angiogenesis defects were rescued by N-terminal sequence of udu mRNA, but not the C-terminal one. To further investigate the domain function in vivo and study the relationship between udu and udub, five CRISPR zebrafish knockout (KO) lines were generated, accompanied with different nonsense mutations in udu and udub. The morphology in udunn11, predicted containing two YY1BD, also had hematopoietic and angiogenic defects. On the other hand, there were no significant differences in udub knockout lines compared with siblings during embryonic development, but there was abnormal mass mortality in udub mutants from 3 month post fertilization (mpf) and their body length is shorter. Through H&E and PCNA stainings, we found that the anatomy and histology of the liver in udub adult fish were abnormal and had lower cell proliferation signals. Finally, we also found the udu RNA expression in udub mutants was down-regulated, which suggests that udub modulates the udu expression. In conclusion, udutu24, udusq3, and udunn11 have similar defects during embryonic development. In contrast, udub mainly affects the development and death of adult fish, suggesting that udu and udub play important roles in embryonic and adult stages, respectively. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65027 |
DOI: | 10.6342/NTU202000512 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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