The phiC31 Phage Integrase for Plastid Engineering in Higher Plants

在模式植物拟南芥中，叶绿体转化效率低下。拟议中的研究将检验这样一个假设，即拟南芥中低效的叶绿体转化是由于叶绿体的同源重组机制在整合外源DNA方面效率低下。通过利用phiC31噬菌体整合酶(INT)的独立于同源重组的系统来提高转化效率，将检验这一假设。整合体的转化将取决于一种受体系的可用性，在该受体系中attB位点已被整合到叶绿体基因组中。叶绿体的转化将包括通过核编码的、以叶绿体为靶标的整合体将attP载体插入attB位点。转化了叶绿体基因组的细胞将通过携带在attP叶绿体载体上的卡那霉素或壮观霉素抗性进行选择。在常规基础上对拟南芥质体基因组进行转化，将使基于转基因的拟南芥遗传筛选能够在细胞核和细胞器之间进行通讯和分子相互作用的调节因子筛选。为拟南芥开发的方法可能适用于油菜(甘蓝型油菜)和蔬菜甘蓝(花椰菜、花椰菜和卷心菜；甘蓝)，这两个物种与拟南芥有关，在北美存在严重的基因流动问题。由于在十字花科植物中，叶绿体不是通过花粉传播的，在拟南芥中开发的方法也可能在农业上应用于控制田间的转基因流动。

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.