A novel approach to plastid transformation utilizes the phiC31 phage integrase
A novel approach to plastid transformation utilizes the phiC31 phage integrase
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DOI：
10.1111/j.1365-313x.2004.02015.x
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发表时间：
2004-03-01
期刊：
影响因子：
7.2
通讯作者：
Maliga, P
中科院分区：
文献类型：
--
作者：
Lutz, KA;Corneille, S;Maliga, P
迄今为止，高等植物中的质体转化是基于通过质体的同源重组机制将外源DNA掺入质体基因组中。我们在此报告了一种替代方法，该方法依赖于 phiC31 噬菌体位点特异性整合酶（INT）对外源 DNA 的整合，介导细菌和噬菌体附着位点（分别为 attB 和 attP）之间的重组。新方法的质体转化取决于受体系的可用性，其中attB位点已通过同源重组整合到质体基因组中。质体转化涉及通过 INT 将 attP 载体插入 attB 位点，并通过选择 attP 质体载体中携带的抗生素抗性来选择转质体克隆。 INT 功能是通过编码质体靶向 INT 的核基因表达或从质体中的非整合质粒瞬时表达 INT 来提供的。使用具有卡那霉素抗性或奇霉素抗性作为选择标记的 attP 载体，两种方法的转化均获得成功。一些稳定核 INT 系的转化效率高达每个轰击样品 17 个独立转化的系。由于该系统不依赖于质体的同源重组机制，我们预计基于 INT 的载体将使质体转化成为同源重组很少产生转质体克隆的物种的常规。
Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.