Identification of Plant Genes Involved in Agrobacterium-Mediated Transformation

本项目的目的是鉴定参与农杆菌介导的遗传转化的植物基因，并利用这些信息提高玉米的遗传转化效率，防止葡萄冠瘤病的发生。农杆菌介导法是目前用于获得转基因植株的最流行的方法。然而，人们对转型过程的许多方面知之甚少。具体地说，人们对宿主基因和蛋白质在农杆菌-植物细胞相互作用中所扮演的角色知之甚少。了解植物在转化过程中的作用，肯定会导致对农艺上重要的物种进行基因操作的新技术，这些物种目前对农杆菌介导的转化具有抵抗力。例如，许多植物可以相对有效地被农杆菌瞬时转化，但它们的稳定转化仍然难以捉摸。很可能这些作物在T-DNA整合到植物基因组方面存在缺陷。对整合过程中涉及的植物基因的了解可能会让科学家设计出策略，在顽固的物种中过度表达这些基因的同源基因，并将它们改变为转化能力。另一方面，在敏感植物中，相同基因的失活将使它们对冠瘤具有抵抗力。冠瘤病影响到许多目标作物物种，其预防对农业社会的许多部门都是重要的。该项目涉及三个实验室(两个在美国，一个在瑞士)的国际合作，并将使用四种方法来识别和表征参与农杆菌介导的转化的植物基因：1)筛选现有的T-DNA标记的拟南芥突变体鼠(抗农杆菌转化)表型。将对这些突变体进行遗传和生化研究，以确定转化过程受阻的具体阶段；2)利用酵母单杂交和双杂交系统来鉴定与由农杆菌转移到植物中的Vir(毒性)蛋白相互作用的植物蛋白。这些VIR蛋白包括VirF、VirB2、VirD2和VirE2；3)利用差异显示、微阵列和消减方法来鉴定在农杆菌介导的转化早期阶段表达上调或下调的植物基因；4)利用反向遗传学来鉴定参与农杆菌介导的转化的拟南芥基因，并利用每个合作实验室开发的特定生物测试来检测由这些基因编码的蛋白质的功能。从我们对拟南芥基因的分析中获得的信息将被用于转基因玉米，使其更容易受到农杆菌介导的转化，并将用于转基因葡萄，使其对冠瘤病具有抗性。交付成果：通过电子邮件发送PI的其他项目信息，网址为：http://www.bio.purdue.edu/courses/gelvinweb/gelvin.html

The objectives of this project are to identify plant genes involved in Agrobacterium-mediated genetic transformation, and subsequently to utilize this information to improve the genetic transformation of maize and to prevent crown gall disease on grape. Agrobacterium-mediated transformation is the most popular method currently in use for generating transgenic plants. However, there are many aspects of the transformation process that are poorly understood. Specifically, little is known about the roles that host genes and proteins play in Agrobacterium-plant cell interactions. Understanding of the plant contribution to the transformation process will certainly result in new technologies for genetic manipulation of agronomically important species that are presently recalcitrant to Agrobacterium-mediated transformation. For example, many plants can be relatively efficiently transformed transiently by Agrobacterium, yet their stable transformation remains elusive. It is likely that these crops are deficient in T-DNA integration into the plant genome. An understanding of the plant genes involved in the integration process may allow scientists to devise strategies to over-express homologues of these genes in the recalcitrant species and alter them to transformation-competence. On the other hand, inactivation of the same genes in susceptible plants will make them crown gall-resistant. Crown gall disease affects numerous target crop species, and its prevention would be important for many sectors of the agricultural community. This project involves an international collaboration of three laboratories (two in the US and one in Switzerland) and will use four approaches to identify and characterize plant genes involved in Agrobacterium-mediated transformation: 1) Screening of existing collections of T-DNA-tagged Arabidopsis mutants for rat (resistant to Agrobacterium transformation) phenotypes. These mutants will be investigated genetically and biochemically to identify the specific stage at which the transformation process is arrested; 2) Utilization of the yeast one- and two-hybrid systems to identify plant proteins that interact with Vir (virulence) proteins transferred to the plant by Agrobacterium. These Vir proteins include VirF, VirB2, VirD2, and VirE2; 3) Utilization of differential display, microarray, and subtraction approaches to identify plant genes whose expression is up- or down-regulated during early stages of Agrobacterium-mediated transformation; 4) Characterization of Arabidopsis genes involved in Agrobacterium-mediated transformation using reverse genetics, and examination of the functions of proteins encoded by these genes using specific biological assays developed in each of the collaborating laboratories. The information gained from our analysis of Arabidopsis genes will be used to genetically modify maize such that it will become more susceptible to Agrobacterium-mediated transformation, and to genetically modify grape such that it will become resistant to crown gall disease. Deliverables: E-mail the PI Additional project information can be found at: http://www.bio.purdue.edu/courses/gelvinweb/gelvin.html