TMV-COAT PROTEIN IN ENGINEERED CROSS-PROTECTION

工程交叉保护中的 TMV 涂层蛋白

• 批准号: 3141280
• 负责人: Roger Beachy
• 金额: $ 11.91万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-02-01 至 1992-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3141280
• 关键词: chimeric proteins; genetic mapping; genetic strain; genetically modified plants; mutagens; mutant; plant virus; point mutation; protein engineering; protein sequence; site directed mutagenesis; tobacco mosaic virus; transfection; transposon /insertion element; virus cytopathogenic effect; virus envelope; virus genetics; virus infection mechanism; virus protein; virus replication

Genetic transformation of plants to constitutively express virus coat protein genes has been used to confer protection against 5 different plant viruses. Recently, we completed the first field tests with tomato plants and documented field level production against tobacco mosaic virus (TMV). However, the cellular and molecular bases for the engineered protection are not known. Recently, we demonstrated that protection probably blocks an early stage in virus infection that prevents uncoating of the virus. A second manifestation of protection is that systemic movement of virus is slowed in these transgenic plants. We have also conclusively demonstrated that the capsid protein (rather than it's mRNA) is responsible for conferring protection (Powell Abel et al., manuscript in preparation). We now propose to carry out in vivo and in vitro experiments to: (1) determine the role of specific amino acid sequences or protein structures in the protection reaction by producing mutants that affect the sequence and length of the coat protein; (2) determine the amino acid sequences (or protein domains) that confer strain specificity to the protection reaction by producing chimeric proteins comprised of segments of two strains that do not cross-protect against each other (i.e., the common strain and legume strains of (TMV); (3) characterize the protection against systemic virus movement and so determine the role of specific amino acid sequences in such protection. These experiments will lead to a more complete understanding of genetically engineered (cross) protection, and enable the formulation of rational approaches to improve and/or extend the protection.

组成型表达病毒的植物遗传转化 外壳蛋白基因已被用来赋予保护免受5 不同的植物病毒 最近，我们完成了第一个领域， 番茄植株试验和记录的田间生产 抗烟草花叶病毒（TMV）。 然而，细胞和 用于工程保护的分子基础是未知的。 最近，我们证明了保护可能会阻止早期的 病毒感染的一个阶段，防止病毒的外壳脱落。 一 保护的第二个表现是， 病毒在这些转基因植物中的传播速度减慢。 我们还 最终证明，衣壳蛋白（而不是它的 mRNA）负责赋予保护作用（Powell Abel等人， 手稿正在准备中）。 我们现在建议在体内进行 和体外实验，以：（1）确定特定的作用 氨基酸序列或蛋白质结构的保护 通过产生影响序列和长度的突变体的反应 （2）确定外壳蛋白的氨基酸序列（或 蛋白质结构域）赋予菌株特异性以保护 通过产生嵌合蛋白质的反应，所述嵌合蛋白质由以下片段组成： 两种菌株不相互交叉保护（即，的 TMV的常见株系和豆类株系;（3）表征 防止系统性病毒移动，从而确定 特定氨基酸序列在这种保护中的作用。 这些 实验将使我们更全面地了解 基因工程（交叉）保护，并使 制定合理的办法，以改善和/或扩大 保护