Identification of Plant Genes Involved in Agrobacterium-Mediated Transformation

农杆菌介导转化涉及的植物基因的鉴定

• 批准号: 9975715
• 负责人: Stanton Gelvin
• 金额: $ 444.08万
• 依托单位: Purdue Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9975715&HistoricalAwards=false
• 关键词: Identification; Plant; Genes; Involved; Agrobacterium

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

本项目的目的是鉴定农杆菌介导的遗传转化中涉及的植物基因，并随后利用这些信息来改进玉米的遗传转化和预防葡萄冠瘿病。农杆菌介导的转化是目前用于产生转基因植物的最流行的方法。然而，人们对转型过程的许多方面知之甚少。具体而言，很少有人知道的作用，宿主基因和蛋白质在土壤杆菌-植物细胞相互作用。了解植物在转化过程中的作用，必将产生新的技术，用于目前对农杆菌介导的转化无效的农艺学上重要的物种的遗传操作。例如，许多植物可以通过农杆菌相对有效地瞬时转化，但它们的稳定转化仍然是难以捉摸的。这些作物可能缺乏T-DNA整合到植物基因组中。对参与整合过程的植物基因的理解可能使科学家能够设计出策略，以过度表达这些基因的同源物，并改变它们的转化能力。另一方面，在易感植物中相同基因的失活将使它们对冠瘿具有抗性。冠瘿病影响许多目标作物物种，其预防对农业社区的许多部门都很重要。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的大鼠（耐农杆菌转化）表型。将对这些突变体进行遗传学和生物化学研究，以确定转化过程被阻止的特定阶段; 2）利用酵母单杂交和双杂交系统来鉴定与通过农杆菌转移到植物的Vir（毒力）蛋白相互作用的植物蛋白。（3）利用差异显示、微阵列和差减法鉴定在农杆菌介导的转化早期表达上调或下调的植物基因; 4）使用反向遗传学表征参与农杆菌介导的转化的拟南芥基因，并使用每个合作实验室开发的特定生物测定法检查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用于遗传修饰玉米，使其对农杆菌介导的转化更敏感，并用于遗传修饰葡萄，使其对冠瘿病具有抗性。附件：通过电子邮件发送PI其他项目信息可在http://www.bio.purdue.edu/courses/gelvinweb/gelvin.html上找到