I.Establishing Yeast as a Model Host to Study Replication of Bamboo Mosaic VirusII. Potential Use of Sporosarcina pasteurii to Promote the Solidification of Reservoir Sludge

• Main Authors: Yu-Ting Huang, 黃郁婷
• Other Authors: Menghsiao Meng
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/cd74qn

碩士 === 國立中興大學 === 生物科技學研究所 === 99 === 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.