Identification of Host Factors Using the Hydrophilic Region and RNA-Dependent RNA Polymerase of Bamboo Mosaic Virus Replicase as Bait in Yeast Two-Hybrid Screen

• Main Authors: HuiChuan Wu, 吳慧娟
• Other Authors: 孟孟孝
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/97157053554241335344

碩士 === 國立中興大學 === 生物科技學研究所 === 91 === Bamboo mosaic virus (BaMV), a member of the potexvirus group, contains a single-stranded, positive sense RNA genome with five open reading frames (ORFs) and infects primarily members of the Bambusoideae, few other monocotyledonous plants and Nicotiana benthamiana. ORF1 encodes a 155-kDa replicase divided into capping enzyme, RNA helicase-like domain, and RNA-dependent RNA polymerase (RdRp) on the order of from N to C termini. Between the first two domains is a highly hydrophilic region (HR) with approximate 110 amino acids, while a proline-rich stretch sits between the latter two domains. To investigate the potential role of the hydrophilic region and search for cellular proteins involved in the viral replication, yeast two-hybrid screening against a leaf cDNA library of N. benthamiana is being employed by using the hydrophilic region and RdRp as bait. At first, the bait plasmids were constructed. RdRp was a well-behaved bait, but the hydrophilic region itself can transactivate the reporter genes (HIS3 lacZ). To avoid the nonspecific activation activity, the hydrophilic region was variously truncated, and finally the one with 33 amino acids deleted at N terminus (HRΔN) was used as bait in yeast two-hybrid screen. In the meanwhile, a leaf cDNA library containing 3.5×104 clones of N. benthamiana was constructed. Using HRΔN as bait to screen the leaf cDNA library, 26 colonies could grow up on minimal medium that lacked histidine (His+) and showed β-galactosidase activity (LacZ+). However, when putative interactors were retrieved and retested the ability of the two-hybrid proteins to interact specifically by retransforming to yeast, both of HIS3 and LacZ reporter genes could not be activated. It meant that all of 26 clones were false positive. This may result from the deletion of the N terminus of HR. On the other hand, 582 double-positive colonies (His+ LacZ+) were obtained when RdRp was used as bait. Using quantitative assay of β-galactosidase activity, 47 clones showed stronger interaction. By retrieving these putative interactors and retransforming to yeast to test the specific interaction, 25 clones can specifically interact with RdRp. Then the nucleotide sequences of these clones were determined and their deduced amino acid were searched in database. As a result, their encoded proteins have homology with four class proteins including photosynthesis related proteins, stress induced proteins, ubiquitin-proteasome pathway related proteins and unknown function proteins. In the future, biochemical assay will have to be performed to confirm these interactions. And how these proteins interacted with RdRp to affect the growth of the host plant or regulate virus replication? They also have to be further investigated.