Establishing the purification system for the host proteins interacting with Tomato leaf curl New Delhi virus movement protein for the functional study of the movement protein on mechanical transmissibility

• Main Authors: Shu-Hui Lin, 林書卉
• Other Authors: Fuh-Jyh Jan
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/75109462957048386696

碩士 === 國立中興大學 === 植物病理學系所 === 102 === Plant-infecting geminiviruses cause considerable crop losses and are threatening commercial crop production worldwide. The virus contains circular single-stranded DNA genome encapsidated in geminate particles. Tomato leaf curl New Delhi virus (ToLCNDV), a geminivirus that severely affects tomato production, contains bipartite genome, designated as DNA-A and DNA-B. DNA-A encodes the coat protein, the replication-associated proteins, and the proteins involved in gene expression. DNA-B encodes the proteins responsible for the virus movement. Generally, most ToLCNDV isolates are not mechanically transmissible to its natural hosts, but a ToLCNDV oriental melon (ToLCNDV-OM), an isolate identified by molecular virology laboratory at National Chung Hsing University in 2007 is mechanically transmissible. Our previous study showed that ToLCNDV-OM movement protein (MP) is the critical factor for its mechanical transmissibility. In this study we established a purification system that combines tissue fractionation by differential centrifugation, surfactant extraction and batch affinity purification. We also found an unidentified Nicotiana protein, P47 that may specifically interact with ToLCNDV-OM MP but not with ToLCNDV-CB MP via this soluble membrane protein complex purification system. Further study on the analysis of P47 by using the liquid chromatography–mass spectrometry is needed to confirm this specific interaction and its significance to the mechanical transmissibility of ToLCNDV. The affinity purification combined with the mass spectrometry is one of the reliable methods for the isolation of the protein complex and the analysis of the interaction patterns can provide hints for the unknown functions of a protein. The purification system established here has also the potential for other membrane protein complex purification.