Analysis of virB genes in Agrobacterium T-DNA transfer

在根癌农杆菌转化植物的过程中，VIRB基因产物很可能介导T-DNA跨细菌和植物细胞膜和细胞壁的运输。这项建议的长期目标是阐明11个VIRB基因产物在T-DNA转移过程中的作用。这些特定的目的将检验以下问题：1)哪些VIRB基因是毒力所必需的？2)VIRB蛋白是否构成T-DNA的运输机制？3)磷酸化是否参与控制或激活VIRB功能？这些问题将结合遗传和生化方法加以解决。首先，将提高VIRB特异性抗体，并分离出每个都缺乏不同单一VIRB基因的根癌农杆菌突变体。然后，这些抗血清和突变体将与细胞分离、免疫电子显微镜、蛋白质交联、定点突变和其他技术结合使用，以鉴定、定位和功能鉴定VIRB基因产物。这一结果将对深入了解T-DNA的运输机制具有直接的意义。在实际应用水平上，对农杆菌VIRB基因功能的表征可能有助于理解大分子跨膜运输的分子机制。

During plant transformation by Agrobacterium tumefaciens the virB gene products most likely mediate T-DNA transport across the bacterial and plant cell membranes and walls. The long-term objective of this proposal is to elucidate the role of the eleven virB gene products in the T-DNA transfer process. The specific aims will examine the following questions: 1) Which virB genes are needed for virulence ? 2) Do VirB proteins compose the T-DNA transport machinery? and 3) Is phosphorylation involved in controlling or activating VirB functions? These questions will be addressed using a combination of genetic and biochemical approaches. First, VirB-specific antibodies will be raised and A. tumefaciens mutants will be isolated that each lack a different single virB gene. These antisera and mutants will then be used in combination with cellular fractionation, immunoelectron microscopy, protein cross-linking, site specific mutagenesis, and other techniques to identify, localize, and functionally characterize the virB gene products. The results will have immediate significance in providing insight into the mechanism of T-DNA transport. On a practical level, characterization of virB gene function in Agrobacterium will likely contribute to understanding the molecular mechanism by which macromolecules are transported across membranes.