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

该项目的目标是确定与农杆菌介导的遗传转化有关的植物基因，然后利用这些信息来改善玉米的遗传转化并防止葡萄染色的冠状病。农杆菌介导的转化是目前用于生成转基因植物的最流行方法。但是，转型过程的许多方面知之甚少。具体而言，关于宿主基因和蛋白质在农业植物 - 植物细胞相互作用中起的作用知之甚少。对植物对转化过程的贡献的理解无疑会导致对农业上重要物种的遗传操纵的新技术，这些物种目前对农杆菌介导的转化是顽固的。例如，许多植物可以通过农杆菌相对有效地瞬时地转化，但它们的稳定转化仍然难以捉摸。这些农作物可能缺乏T-DNA整合到植物基因组中。对综合过程中涉及的植物基因的理解可能会使科学家能够制定策略过表达这些基因在顽固物种中的同源物，并将其改造成转化能力。另一方面，易感植物中相同基因的灭活将使它们抗胆汁。冠状疾病会影响许多目标作物物种，其预防对农业界的许多部门都很重要。该项目涉及三个实验室的国际合作（在美国和一个在瑞士）进行的，并将使用四种方法来识别和表征与农杆菌介导的转化有关的植物基因：1）筛选现有的T-DNA-Tagged Abledopsis集合的大鼠（对农业对农业的抗体转化）的现有收集。这些突变体将在遗传和生物化学上进行研究，以确定逮捕转化过程的特定阶段。 2）利用酵母单和两杂交系统来鉴定与农杆菌转移到植物的VIR（毒力）蛋白相互作用的植物蛋白。这些VIR蛋白包括VIRF，VirB2，Vird2和Vire2； 3）利用差分显示，微阵列和减法方法来鉴定在农杆菌介导的转化的早期阶段表达上或下调的植物基因； 4）使用反向遗传学的拟南芥基因进行了参与农杆菌介导的转化的表征，并使用每个协作实验室中开发的特定生物学测定法对这些基因编码的蛋白质功能进行了检查。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，以使其更容易受到农杆菌介导的转化的影响，并遗传改变葡萄，以使其对冠状病具有抵抗力。可交付物：电子邮件PI其他项目信息可以找到：http：//www.bio.purdue.edu/courses/gelvinweb/gelvin.html