Molecular Analysis of the TMV-Movement Protein, A Virulence Factor

植物病毒通过一种或多种病毒基因产物的表达，通过未知的机制在宿主体内传播。由于不在宿主中传播的病毒通常不具有致病性，因此在接种植物中的移动能力构成了毒力功能。然而，蛋白质的性质以及蛋白质发挥作用的分子和细胞机制尚未确定。最近，本实验室通过利用转基因植物组成性地表达编码30 kDa蛋白的嵌合基因，证明烟草花叶病毒（TMV）的30 kDa蛋白负责增强TMV的细胞间运动。该系统将用于：1)纯化和表征tmv - 30kda蛋白（这里称为运动蛋白，MP）；2)制备抗TMV-MP的多克隆抗体；3)在转基因和感染tmv的烟草植株中定位MP；4)通过位点定向和随机诱变的方法，对传染性TMV克隆cDNA进行基因定位，定位MP的功能域/氨基酸，挽救表达MP的转基因植物上的缺陷突变体。通过在表达30kda蛋白的转基因植株中确定运动缺陷的病毒突变体的特征，本研究的结果将有助于了解TMV- 30kda蛋白在烟草植株中增强TMV运动的功能。

Plant viruses move throughout their hosts by unknown mechanisms that involve expression of one or more viral gene products. Since viruses that do not spread in their hosts are generally not pathogenic, the capacity for movement in the inoculated plant constitutes a virulence function. However, the nature of the protein(s) and the molecular and cellular mechanisms by which the proteins function are not yet determined. Recently this laboratory demonstrated that the 30 kDa protein of Tobacco Mosaic Virus (TMV) is responsible for potentiating cell-to-cell movement of TMV by using transgenic plants that constitutively express a chimeric gene encoding the 30 kDa protein. This excellent system will be used to: 1) purify and characterize the TMV-30 kDa protein (referred to here as the movement protein, MP); 2) to generate polyclonal antibodies against the TMV-MP; 3) to localize the MP in transgenic and TMV-infected tobacco plants; and 4) to genetically map the functional domains/amino acids of the MP by site directed and random mutagenesis of an infectious cloned cDNA of TMV, rescuing defective mutants on transgenic plants that express the MP. By characterizing the virus mutants that are defective in movement, as determined by ability to move in the transgenic plants that express the 30 kDa protein, the results of this research will lead to an understanding of the function of TMV-30 kDa protein in potentiating the movement of TMV in tobacco plants.