In vitro Reconstruction and Biochemical Characterization of the Agrobacterium T-DNA Transfer Process

这项拟议的研究将有助于进一步了解细胞转化。对数据的研究使用了整个农杆菌细胞来鉴定几种(VIR)蛋白质产物，这些蛋白质产物对于生产可转移的DNA拷贝T-链是必不可少的。拟议的实验将使用主要的生化方法来分析T链的合成，并在体外使用纯化的成分将其加工成T链蛋白质复合体(T复合体)。具体地说，这些研究将(1)使用DNA底物和纯化的VIR蛋白在体外重建T链合成。如有必要，可使用市售的酶(DNA聚合酶、拓扑异构酶)或农杆菌全细胞提取物来补充反应。(2)将进行检测，以确定迄今表征(并在体外组装)由T-链、VirE2和VirD2组成的T-复合体是否能够转化植物原生质体。随着T-复合体的其他蛋白质被鉴定出来，它们将被添加到T-链-VirD2-VirE2复合体中，以测试它们在促进植物细胞转化方面的作用。(3)增强T-复合体转移的其他蛋白质将由它们与携带共价结合的VirD2或VirE2的柱子的亲和力以及最近开发的一种介导整个农杆菌细胞T链输出的蛋白质的分析来定义。(4)T链合成和加工所需蛋白质的功能域将通过分析缺失突变体或蛋白水解酶产生的这些蛋白质的活性亚片段来确定。(5)用一种灵敏的外源DNA整合实验来确定T-复合体是否具有整合酶功能。拟议的实验与农杆菌-植物细胞相互作用的生物学和DNA转移技术有关。在自然界中，农杆菌将特定的DNA片段T-DNA转移到植物细胞核中，在那里稳定整合后，遗传物质在植物细胞中表达。农杆菌的这一特性促使人们对转移的要求进行了深入的研究，并导致了能够将任何感兴趣的DNA转移到植物细胞而不干扰植物正常生长和分化的载体的设计。虽然农杆菌现在可以常规地用作载体，将DNA转移到许多植物细胞，而不需要了解涉及的潜在机制，但重要的是了解系统的生物学，以便在使用系统的载体特性方面取得进展。这项研究将研究DNA转移过程中涉及的详细步骤，以便改进允许将DNA定向转移到植物细胞的方法。

The proposed research will allow for a further understanding of cell transformation. Studies to data have used whole Agrobacterium cells to identify several (vir) protein products essential for the production of a transferable DNA copy, the T-strand. The proposed experiments will use predominantly biochemical approaches to analyze T-strand synthesis and processing into a T-strand-protein complex (T- complex) using purified components in vitro. Specifically, these studies will (1) reconstruct T-strand synthesis in vitro using a DNA substrate and purified vir proteins. If necessary, commercially available enzymes (DNA polymerases, topoisomerases) or whole cell extracts from Agrobacterium will be used to supplement the reaction. (2) Assays will be carried out to determine whether the T-complex characterized to date (and assembled in vitro) consisting of T-strands and VirE2 and VirD2 is capable of transforming plant protoplasts. As other proteins of the T-complex are identified they will be added to the T-strand-VirD2-VirE2 complex to test their role in enhancing plant cell transformation. (3) Additional proteins that potentiate T-complex transfer will be defined by their affinity to columns carrying covalently bound VirD2 or VirE2 and by a recently developed assay for proteins that mediate export of T-strands from whole Agrobacterium cells. (4) The functional domains of proteins required for T-strand synthesis and processing will be defined by assaying for activity subfragments of these proteins produced by deletion mutants or proteolytic digestion. (5) By using a sensitive in vitro assay for integration of exogenous DNA it will be determined if the T-complex has an integrase function. The proposed experiments are relevant to the biology of the Agrobacterium-plant cell interaction and to DNA transfer technology. In nature, Agrobacterium transfers a specific DNA segment, the T-DNA, into a plant nucleus where, following stable integration, the genetic material is expressed in the plant cell. This property of Agrobacterium has prompted an intense investigation of the requirements for transfer and has led to the design of vectors capable of transferring any DNA of interest to plant cells without interfering with normal plant growth and differentiation. While Agrobacterium can now be used routinely as a vector to transfer DNA to many plant cells without the need to understand the underlying mechanisms involved, it is important to understand the biology of the system so that advances in the use of the vector properties of the system can be made. This research will investigate the detailed steps involved during the DNA transfer process so that improved methods to allow directed DNA transfer to plant cells can be achieved.