Berger P H, Hunt A G, Domier L L, Hellmann G M, Stram Y, Thornbury D W, Pirone T P
Department of Plant Pathology, University of Kentucky, Lexington, 40546-0091.
Proc Natl Acad Sci U S A. 1989 Nov;86(21):8402-6. doi: 10.1073/pnas.86.21.8402.
Helper component (HC) is a virus-encoded nonstructural protein that is required for transmission of potyviruses by their aphid vectors. As a prelude to studies on the molecular basis of HC activity, a cDNA clone (pPB-3) was constructed that contained the first three cistrons (34 kDa-HC-42 kDa) of the RNA genome of the potyvirus tobacco vein mottling virus, the first six nucleotides of the adjacent cylindrical inclusion body protein cistron, and a synthetic translation termination codon. This construction was introduced into tobacco cells via a Ti plasmid-based vector. Northern blot analysis of transgenic plants demonstrated the presence of an RNA of the size expected from the construction of pPB-3, and Western blot analysis revealed the presence of a protein that comigrated with authentic HC, indicating that the proteolytic activity necessary to produce mature-sized HC was encoded by pPB-3. The HC produced in the transgenic plants was demonstrated to be active in a virus transmission bioassay with aphids.
辅助成分(HC)是一种病毒编码的非结构蛋白,是马铃薯Y病毒通过蚜虫介体传播所必需的。作为对HC活性分子基础研究的前奏,构建了一个cDNA克隆(pPB - 3),它包含马铃薯Y病毒烟草脉斑驳病毒RNA基因组的前三个顺反子(34 kDa - HC - 42 kDa)、相邻柱状内含体蛋白顺反子的前六个核苷酸以及一个合成的翻译终止密码子。通过基于Ti质粒的载体将该构建体导入烟草细胞。对转基因植物的Northern印迹分析表明存在与pPB - 3构建体预期大小相符的RNA,Western印迹分析显示存在与天然HC迁移率相同的蛋白质,这表明产生成熟大小HC所需的蛋白水解活性由pPB - 3编码。在转基因植物中产生的HC在蚜虫介导的病毒传播生物测定中被证明具有活性。