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標題: | 尋找與計畫性細胞死亡相關之新基因 Identifying new Programmed Cell Death Genes in C.elegans |
作者: | Pin-Chieh Chiang 江品潔 |
指導教授: | 吳益群(Yi-Chun Wu) |
關鍵字: | 計畫性細胞死亡,線蟲,單一核甘酸多型性,RNA干擾,細胞屍體, programmed cell death,C. elegans,Single-nucleotide polymorphism(SNP),RNA interference,cell corpse, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 計畫性細胞死亡是多細胞生物發育與維持生理恆定的重要機制,主要分為決定、執行、吞噬、分解四個過程。執行階段主要由四個重要的基因EGL-1、CED-9、CED-4、CED-3所調控。然而,EGL-1的表現如何被調控以及CED-3所作用的受質有哪些進而影響計畫性細胞死亡,目前的研究並不清楚。我們以線蟲為材料,利用線蟲雌雄同體在發育的特定時期會有特定的細胞進行計畫性細胞死亡的特性,分別以正向遺傳學和反向遺傳學為策略,試圖能夠找到一些參與在計畫性細胞死亡的新基因。
在正向遺傳篩選策略的部分,研究室先前的成員觀察到當grp-1和egl-1、ced-4、ced-3雙重突變時會導致尾巴型態異常,但他們分別單獨突變時卻沒有。因此 以一個grp-1突變的敏感化背景,分離出許多個會導致尾巴型態異常的突變。在這個篩選下,CH2126被分離出來,我進一步分析顯示這個突變株有23%的尾巴型態異常,在胚胎1.5摺時期的有0.5倍的細胞屍體下降。除此之外,約有一半的蟲會有多出來的AVM、PVM觸覺神經細胞。利用細胞屍體的性狀搭配單一核單酸多型性做定位我發現這個突變株的性狀可能由雙重突變tp28(位於X染色體)、tp190(位於II染色體)所造成。進一步遺傳分析,發現tp190就是造成多出來的AVM、PVM觸覺神經細胞和尾巴型態異常性狀的突變。 在反向遺傳篩選策略的部分,過去學者找到一些可能參與在果蠅計畫性細胞死亡的基因。我們與NHRI的陳俊宏老師合作,利用資料庫的比對,首先找到這些基因在線蟲的相對應基因,再以RNA干擾術降低這些基因表現並觀察。我們發現大部分基因在線蟲生殖細胞的計畫性細胞死亡可能也扮演功能,而hum-5和cbd-1可能同時也參與在體細胞計畫性死亡。 我的正、反向遺傳研究結果顯示,尚有一些先前未知的基因可能同時影響線蟲體細胞和生殖細胞之計畫性細胞死亡,而他們的功能可能在演化上具有保守性。 Programmed cell death is important for development and homeostasis for multicellular organisms. The process for programmed cell death consists of four steps: decision, execution, engulfment, and degradation. In C. elegans, the execution step is regulated by EGL-1/BH-3 only protein, CED-9/Bcl-2, CED-4/Apaf-2 and CED-3 / caspase. However, the regulators of egl-1 transcription, and the identities of CED-3 substrates are not clear. To identify new players regulating programmed cell death, we performed both forward and reverse genetic screens. The forward genetic screen was based on a previous observation that the double mutants of grp-1 and egl-1, ced-3 or ced-4 show a synthetic tail defect, but their respective single mutants do not. Thus, several mutations that result in a tail defect in the sensitized grp-1 background were isolated. CH2126, a noteworthy strain with 23% penetrance of the tail defect, was shown to be grp-1-dependent. In addition, this strain has a decreased cell corpse number at the1.5-fold embryonic stage and extra touch neurons AVM and PVM in larvae. Through SNP mapping, the decreased cell corpse phenotype was found to have been caused by double mutations: tp28 (on chromosome X) and tp190 (on chromosome II). Further genetic analysis showed that the extra touch neuron phenotype and the tail defect were the result of the tp190 but not tp28 mutation. In a reverse genetic approach, we focused on some candidate genes whose homologues in Drosophila have been indicated to be important for programmed cell death by RNA interference. Through collaboration with Dr. Chun-Hong Chen in NHRI, we set out to test if any of these genes may have a conserved role in cell death in C. elegans. By RNA interference and subsequent cell corpse analysis, our data suggest that some of these genes may play a role in germ cell death and that hum-5 and cbd-1 may also affect somatic cell deaths in C. elegans. Both forward and reverse genetics approaches show that additional components are involved in programmed cell death in C. elegans and their functions may be conserved in multicellular organisms. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15914 |
全文授權: | 未授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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