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標題: | 降解體成員協同定位oskar 核醣蛋白複合體於卵母細胞後端 Processing body components cooperate to assist the posterior localization of oskar mRNP complex in the oocyte |
作者: | Chu-Ya Cheng 鄭竹雅 |
指導教授: | 周子賓(Tze-Bin Chou) |
關鍵字: | 降解體,卵母細胞發育, oskar,dDcp2,dGe-1, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 果蠅體軸的發育在胚胎發育時期的時候開始建立,一些特定的母源核醣核酸(maternal mRNA)在護理細胞(nurse cell)中做好後會送到卵母細胞(oocyte)內,並且會因微管(microtubule)極性的分布而座落在不同的特定區域。Osk mRNA在護理細胞中做出來後,在微管的動力蛋白(motor protein),Kinesin,的幫助下,藉由微管的運輸送到卵母細胞的後端。是決定腹部體軸發育及形成pole plasm的重要蛋白質。
果蠅去頭蓋蛋白質2(dDcp2)及dGe-1都為組成降解體(P body)的成員之一,之前的研究都著重在他們降解核醣核酸的功能上,並指出這三種蛋白質彼此之間都有交互作用。我們在免疫螢光染色圖中也看見dGe-1及果蠅去頭蓋蛋白質2兩者在卵中的分布位置相近,且有部分重疊在一起。在免疫共沉澱法中也證實這兩者間確實如其他物種一般是有交互作用的。在實驗室之前的研究中顯示果蠅去頭蓋蛋白質2及dGe-1兩種蛋白質在果蠅的oogenesis當中,會對細胞骨架(cytoskeleton)造成一定程度的影響,並連帶影響到osk mRNP複合體的座落,但對於影響的機制目前並不清楚。我們進一步在果蠅中過度表現果蠅去頭蓋蛋白質2或dGe-1,發現在部分的卵中,於後端定位的Osk 蛋白質的量比對照組相對上升,代表果蠅去頭蓋蛋白質2及dGe-1兩者可能都有參與在osk mRNP複合體的座落上。 osk mRNP複合體被運送到卵母細胞後端需要微管及Kinesin的幫忙,而蛋白質被轉譯出來後要定位在後端則需要微絲(actin)及一些微絲相關的蛋白質幫助。為了更了解這兩個蛋白質是如何影響到osk的座落,我們想了解dGe-1及dDcp2可能藉由哪些蛋白質的幫忙共同參與在osk mRNA的運輸上。Kinesin heavy chain (KHC)和Staufen(Stau)為osk mRNP複合體的成員之一,而Dmoe則是和微絲相關的蛋白質。利用免疫共沉澱法觀察dDcp2、dGe-1和KHC、Stau及Dmoe之間的關係。結果顯示,dDcp2和Dmoe及KHC之間是有交互作用的。 因此我們推論果蠅去頭蓋蛋白質2及dGe-1在卵母細胞皮層(oocyte cortex)當中互相會有交互作用,透過影響到不同的蛋白質同時去影響osk mRNP複合體的運輸及Osk蛋白質的定位。另外,我們認為果蠅去頭蓋蛋白質2及dGe-1影響微管組成的這個功能在演化上是具有保留性的。 The Drosophila axes are set up during oogenesis. Most maternal mRNA are made in the nurse cells, and then transported to the oocyte. Specific mRNAs are transported to specific areas by plus or minus end directed microtubule motors. Osk protein is important in determining the posterior pattern, and forming the pole plasm. The osk mRNA is transported with the osk mRNP complex to the posterior pole, and is done by kinesin, a plus end directed microtubule motor. dDcp2 and dGe-1 are both components of the processing body. Their interaction, and function on the degradation mRNAs have been well characterized in Arabidopsis. In our study, the distribution pattern of dDcp2 and dGe-1 in Drosophila oocyte are similar, and they partially colocalize. Through co-immunoprecipitation, we showed that the interaction between dDcp2 and dGe-1 in Drosophila is like in other species. Previous findings in our lab showed that both dDcp2 and dGe-1 are important for microtubule organization in mid-oogenesis, and thus play an important role in transporting osk mRNP complex to the posterior pole of the oocyte. At the present, the role of dDcp2 and dGe-1 on the transportation of osk mRNP complex is poorly understood. We overexpressed dDcp2 or dGe-1 in Drosophila germ line cell, and found that Osk proteins increase in some oocytes’ posterior pole. This suggests both dDcp2 and dGe-1 are involved in osk mRNP complex localization. Transportation of the osk mRNP complex is microtubule and Kinesin dependent, while anchorage is dependent on some actin associate proteins. To understand how these proteins affect the localization of osk mRNA, we examined the interaction among dDcp2, dGe-1, KHC, Stau and Dmoe by co-immunoprecipitation. The results show that KHC and Dmoe interact with dDcp2. Our results suggest that dDcp2 and dGe-1 cooperate to affect osk mRNP complex localization through other intermediate proteins related to osk mRNA transport. Besides, the function of dDcp2 on affecting microtubule organization may be evolutionarily conserved. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15811 |
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顯示於系所單位: | 分子與細胞生物學研究所 |
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