TMV-COAT PROTEIN IN ENGINEERED CROSS-PROTECTION

工程交叉保护中的 TMV 涂层蛋白

• 批准号: 3141284
• 负责人: Roger Beachy
• 金额: $ 12.91万
• 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/3141284
• 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.

植物的遗传转化以组成性表达病毒 外壳蛋白基因已经被用来提供对5 不同的植物病毒。最近，我们完成了第一个领域 对番茄植株进行测试并记录田间水平的生产 抗烟草花叶病毒(TMV)。然而，蜂窝和 这种工程保护的分子基础尚不清楚。 最近，我们证明了保护可能会阻止早期的 病毒感染的阶段，防止病毒脱壳。一个 保护的第二个表现是全身运动 病毒在这些转基因植物中变慢了。我们还有 最终证明衣壳蛋白(而不是它的 M RNA)负责授予保护(Powell Abel等人， 正在准备中的手稿)。我们现在建议在体内进行 并在体外实验中：(1)确定特异性的作用 保护中的氨基酸序列或蛋白质结构 通过产生影响序列和长度的突变体进行反应 (2)测定氨基酸序列(或 蛋白质结构域)，赋予菌株特异性以保护 通过产生由多个片段组成的嵌合蛋白进行反应 两个不相互交叉保护的菌株(即 烟草花叶病毒(TMV)的普通株系和豆科株系；(3)鉴定 对系统性病毒移动的保护，因此决定了 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理解 基因工程(交叉)保护，并使 制定合理的方法来改进和/或扩大 保护。