The phiC31 Phage Integrase for Plastid Engineering in Higher Plants

用于高等植物质体工程的 phiC31 噬菌体整合酶

• 批准号: 0319958
• 负责人: Pal Maliga
• 金额: $ 20万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0319958&HistoricalAwards=false
• 关键词: phiC31; Phage; Integrase; Plastid; Engineering

Plastid transformation is inefficient in the model plant species Arabidopsis thaliana. Proposed research will test the hypothesis that inefficient plastid transformation in Arabidopsis is due to the inefficiency of the plastid's homologous recombination machinery to incorporate foreign DNA. Increased transformation efficiency by a system independent of homologous recombination, utilizing the phiC31 phage integrase (INT), will test this hypothesis. Plastid transformation by INT will depend on the availability of a recipient line in which an attB site has been incorporated in the plastid genome. Plastid transformation will involve insertion of an attP vector into the attB site by a nuclear-encoded, plastid-targeted INT. Cells with transformed plastid genomes will be selected by kanamycin or spectinomycin resistance carried in the attP plastid vector. Transformation of the Arabidopsis plastid genome on a routine basis will allow plastid transgene-based genetic screens for factors mediating communication and molecular interactions between the nuclear and organellar compartments. Methods developed for Arabidopsis are likely to be applicable to oilseed rape (Brassica napus) and vegetable brassicas (broccoli, cauliflower and cabbage; Brassica oleracea), species related to Arabidopsis with significant gene-flow problems in North America. Since plastids are not transmitted by pollen in Brassicaceae, the methods developed in Arabidopsis may also find agricultural applications to control transgene flow in the field.

质体转化在模式植物拟南芥中是低效的。拟进行的研究将检验这一假设，即拟南芥中低效的质体转化是由于质体的同源重组机制不能有效地整合外源DNA。利用phiC 31噬菌体整合酶（INT），通过不依赖于同源重组的系统提高转化效率，将检验这一假设。通过INT的质体转化将取决于其中attB位点已并入质体基因组中的受体系的可用性。质体转化将涉及通过核编码的、质体靶向的INT将attP载体插入attB位点。将通过attP质体载体中携带的卡那霉素或壮观霉素抗性来选择具有转化质体基因组的细胞。常规基础上的拟南芥质体基因组的转化将允许质体转基因为基础的遗传筛选因子介导的通信和细胞核和细胞器室之间的分子相互作用。为拟南芥开发的方法可能适用于油菜（甘蓝型油菜）和蔬菜芸苔属（花椰菜，花椰菜和卷心菜;甘蓝），与拟南芥相关的物种在北美有显着的基因流问题。由于质体不是通过拟南芥科的花粉传播的，因此在拟南芥中开发的方法也可以在农业上应用于控制田间的转基因流动。