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標題: 以竹嵌紋病毒非複製酵素的蛋白質及菸草甲基轉移酶 (NbMTS1) 作為釣餌蛋白質利用酵母菌雙雜交法尋找菸草中具交互作用之蛋白質
Searching the Proteins Interacted with the Non-replicase Proteins of Bamboo Mosaic Virus and the Methyltransferase of Nicotiana benthamiana (NbMTS1) by Yeast Two-hybrid Screen
作者: 任家賢
Ren, Jia-xian
關鍵字: Bamboo mosaic virus;竹嵌紋病毒;Yeast Two-hybrid Screen;酵母菌雙雜交法
出版社: 生物科技學研究所
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竹嵌紋病毒 (Bamboo mosaic virus, BaMV) 為單股正意RNA病毒,基因體含有五個轉譯架構 (open reading frames, ORFs),ORF1可轉譯出複製酵素,其餘ORFs轉譯出非複製酵素的蛋白質。ORF2、ORF3及ORF4為三個互相重疊的基因組成,分別轉譯出的蛋白質 (triple gene block proteins, TGBp1、TGBp2、TGBp3) 與病毒在寄主細胞間之移動有關,ORF5轉譯出病毒包被所需的外鞘蛋白質 (coat protein, CP)。關於這些非複製酵素的蛋白質是否與寄主蛋白質參與交互作用,目前尚不清楚。此外,本實驗室已證實菸草中的一個甲基轉移酶 (methyltransferase in Nicotiana benthamiana, NbMTS1) 與病毒的RNA聚合酵素 (RNA-dependent RNA polymerase, RdRp) 具有交互作用,可抑制病毒感染的能力,由於不排除NbMTS1是透過影響細胞之生理機能而導致病毒複製量下降的原因,因此尋找與NbMTS1有交互作用的寄主蛋白質,有助於釐清NbMTS1抑制病毒複製的詳細機制。本研究為尋找在病毒感染過程中可能與TGBp及CP產生交互作用的寄主蛋白質,以及尋找與NbMTS1具交互作用的蛋白質,因此以TGBp、CP及NbMTS1作為釣餌蛋白質 (bait protein),利用酵母菌雙雜交篩選法 (yeast two-hybrid screen),從菸草葉子的cDNA基因庫中尋找能與釣餌蛋白質交互作用的蛋白質,為了確保完整篩選到所有的cDNA基因庫,每個釣餌蛋白質至少分別進行三重複以上大規模酵母菌轉型作用來篩選cDNA基因庫。TGBp3無法在酵母菌中表現融合蛋白質,因此無法作為此系統的釣餌蛋白質。以TGBp1、TGBp2及NbMTS1作為釣餌蛋白質來篩選菸草葉子cDNA基因庫時,篩選出的質體於再次確認後皆為偽陽性。另一方面,以CP作為釣餌蛋白質時,將篩選到生長在缺乏組胺酸培養基中的菌落,利用β-galactosidase活性測試,篩選出活性較強之轉殖株;經由再次確認質體所轉譯出的蛋白質與CP具有專一性的交互作用。將這些質體定序之後,與蛋白質資料庫比對,發現篩選到的蛋白質為病毒本身的CP,且N端已去除30個胺基酸,顯示竹嵌紋病毒之缺乏N端30個胺基酸CP,不會影響CP間的交互作用。

Bamboo mosaic virus (BaMV), a plus-sense RNA virus, has a genome that consists of five open reading frames (ORFs). ORF1 encodes the viral replicase proteins. ORF2, ORF3, and ORF4 encode triple gene block proteins respectively, (TGBp1, TGBp2, and TGBp3) which are involved in viral movement. ORF5 encodes the viral coat protein (CP). To date, the interaction between the non-replicase proteins and host proteins is not clear yet. The previous study had evidenced the interaction between a putative methyltransferase (NbMTS1) of Nicotiana benthamiana and RNA-dependent RNA polymerase domain (RdRp domain), and the NbMTS1 can inhibit the viral infection, suggesting that it may be a part of the defense mechanism in plant. Since that NbMTS1 can decrease the viral replication, we wondered whether the inhibitory effect is mediated through interaction with other cellular protein. To investigate the host proteins which may interact with CP or TGBp, the TGBp, CP, and NbMTS1 was used as bait proteins in yeast two-hybrid screen to find out the interactive proteins from a leaf cDNA library of N. benthamiana. Each bait protein was executed large-scale library transformation for three times respectively to confirm that we can select all of cDNA library. At first, we used TGBp3 as the bait protein, but it could not be expressed in the yeast. Then we used TGBp1, TGBp2, and NbMTS1. The result of the selected plasmids was all false positive. Finally, we chose CP to be the bait protein. We used some colonies which grew in the absence of histidine medium, and then tested them by β-galactosidase activity assay to select out some stronger interaction. After double checked, we transformed the plasmids to the yeast so that the proteins could interact with CP specifically. We did a DNA sequencing comparison in NCBI BLAST and found that thee proteins were truncated CP; moreover, their N-terminal had already deleted 30 amino acids. This shows the CP-CP interaction would not be affected when the viral CP lacked of N-terminal 30 amino acids.
其他識別: U0005-0202201019545200
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