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標題: I. 建立竹嵌紋病毒於酵母菌中之複製系統 II. 巴氏芽孢桿菌對水庫淤泥固結的可行性探討
I.Establishing Yeast as a Model Host to Study Replication of Bamboo Mosaic Virus II. Potential Use of Sporosarcina pasteurii to Promote the Solidification of Reservoir Sludge
作者: 黃郁婷
Huang, Yu-Ting
關鍵字: Bamboo mosaic virus
yeast screen system
Sporosarcina pasteurii
Microbial-induced calcium precipitation
Reservoir sludge
出版社: 生物科技學研究所
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摘要: 第一部分 : 植物病毒為絕對寄生的微生物,由於病毒基因體大小的限制,導致病毒本身所轉譯的蛋白是有限的,因此在病毒複製時,需要依賴宿主因子的幫助,但是在植物體內研究病毒與宿主之間的交互作用並不容易。由於酵母菌僅有約6000個左右的基因,更有超過60 %的基因已被定義,從基因體或蛋白質體的觀點來看,它是一個很好的模式宿主,適合用來研究病毒與宿主因子之間交互作用的實驗系統。本實驗的目的欲建立竹嵌紋病毒於酵母菌中的複製系統。竹嵌紋病毒是一支隸屬於Flexividiae科Potexvirus屬的正股RNA病毒,含有單股6.4 kb的RNA,其基因體包含5’端甲基帽,五個轉譯區(ORFs)及3’端的poly(A)尾部。首先,我們將glyceraldehydes 3-phosphate dehydrogenase (GAP) promoter置換掉酵母雙雜交用之載體pHybLex/Zeo中的promoter,再把竹嵌紋病毒的基因置入GAP promoter之後,並轉型到酵母菌中進行蛋白質的表現。接著主要使用西方墨點法偵測鞘蛋白質來觀察竹嵌紋病毒在酵母菌中的複製情況。結果顯示,在酵母菌INVSc1、L40、BY4739、BY4741中,均偵測到外鞘蛋白的訊號,而在酵母菌BY4741中,竹嵌紋病毒複製酵素GDD motif 的突變使鞘蛋白可被偵測到的量很微弱,暗示著竹嵌紋病毒能在酵母菌BY4741中複製並轉譯蛋白質,是與病毒複製酵素的能力有關。 第二部分 : 水庫蓄水量與經濟發展息息相關,快速的清淤並確保水庫安全與壽命,是全世界關注的議題,但水庫淤泥顆粒的細小 (< 0.01 mm) 使得淤泥的清運、儲存與再利用難以達成。國際研究報告已經提出Sporosarcina pasteurii (Bacillus pasteurii) 可以分解尿素,產生鹼性環境並提供二氧化碳與鈣離子產生碳酸鈣結晶,填補晶粒之間的空隙。因此本實驗欲利用這樣概念來增加水庫淤泥的粒徑,解決水庫淤泥清運不易的困擾。首先將S. pasteurii培養進行調整測試並發現魚骨粉與雞糞肥可作為適合的經濟培養基,而且本實驗也找到台灣本土之S. pasteurii相似菌株Sporosarcina sp. CN3及Sporosarcina sp. CN6。在S. pasteurii固結淤泥實驗方面,發現高濃度氯化鈣可以促進淤泥的固結效果,並利用氣舉式模式,增加深水區的空氣通透量,擴大S. pasteurii應用於沉澱池中固結水庫淤泥的可能性,也觀察水庫淤泥在掃描式電子顯微鏡和X-ray粉末晶體繞射,發現淤泥顆粒上有碳酸鈣結晶的產生。
Part 1 : Interactions between virus-encoded proteins and host factors are crucial for virus replication in host. On the other hand, the interactions may initiate host defense mechanisms counteracting the viral accumulation. However, identifying such interactions between plant virus and its host is a time-consuming and tedious job. Yeast is a great model system if the studied virus could replicate in yeast from the genome-wide and proteomic viewpoint, because it contains only about 6000 genes, of which over 60% have been characterized. In this study, I tried to establish a replication system of Bamboo mosaic virus (BaMV) in yeast. BaMV is a positive-sense RNA virus belonging to the genus of Potexvirus. The 6.4-kb genome contains five open reading frames (ORFs), a 5’ methyl cap, and a 3’ poly(A). To achieve the goal, the entire cDNA of BaMV was placed downstream the glyceraldehydes 3-phosphate dehydrogenase (GAP) promoter in a yeast episomal vector derived from pHybLex/Zeo (Invitrogen), and the plasmid was named pHGB. pHGB was then transformed into Saccharomyces cerevisiae, INVSc1、L40、BY4739、BY4741. The ability of BaMV replication in yeast was monitored by the expression of coat protein (CP) using Western blotting assay. When GDD motif of RdRp activity site was deleted at pHGB, CP couldn’t be detected in BY4741. Compare to the mutant and the wile type, CP could be accumulated in yeast only when functional BaMV replicase existed. In the future, the replication of BaMV in yeast will be further confirmed and then the host factor-hunting experiment can be carried out. Part 2 : Reservoir storage capacity affects economic development very significantly. Keeping the reservoir security storage level is a worldwide issue; however the reservoir sludge is so small that the precipitation is almost impossible. The previous studies showed that Sporosarcina pasteurii (Bacillus pasteurii) could decomposed urea to produce carbon dioxide and alkaline environment, providing the crystallization of calcium carbonate and fill the gap between the grains. Therefore, this study intended to use this concept to increase the size of the reservoir sludge. We optimized the S. pasteurii growth condition, and found the S. pasteurii analogous organism, Sporosarcina sp. CN3 and Sporosarcina sp. CN6 in Taiwan. For the consolidation experiments of the sludge in S. pasteurii, I found that high concentrations of calcium chloride (250 mM) can promote the efficiency of consolidation. In the air-lift model experiments, high air permeability rising the opportunity of S. pasteurii-mediated the solidification of reservoir sludge in deep water. Furthermore , it was found that the crystallization of calcium carbonate on treated reservoir sludge was increased in the scanning electron microscope and X-ray powder diffraction.
其他識別: U0005-2106201115525000
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