Kittiwongwattana Chokchai, Lutz Kerry, Clark Mark, Maliga Pal
Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA.
Plant Mol Biol. 2007 May;64(1-2):137-43. doi: 10.1007/s11103-007-9140-4. Epub 2007 Feb 9.
Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.
标记基因对于选择性扩增罕见的转化质体基因组拷贝以获得遗传稳定的转质体植物至关重要。然而，当获得同质性植物时，标记基因就变得不再必要。在此我们报道了通过phiC31噬菌体位点特异性整合酶（Int）切除质体标记基因，该整合酶介导细菌（attB）和噬菌体（attP）附着位点之间的重组。我们在一个两步过程中测试了标记基因的切除。首先，我们用pCK2载体转化烟草质体基因组，其中壮观霉素抗性（aadA）标记基因两侧带有方向合适的attB和attP位点。在没有Int的情况下，转化的质体基因组是稳定的。然后我们用一个编码靶向质体的Int的基因转化细胞核，这导致了高效的标记基因切除。不含aadA标记的Nt-pCK2-Int植物对草丁膦除草剂具有抗性，因为pCK2质体载体还携带一个bar除草剂抗性基因，由于其启动子的选择，导致淡黄色-金色（金黄色）表型。本文报道的Int介导的标记切除是目前使用的CRE/loxP质体标记切除系统的一种替代方法，并扩展了可用于操纵质体基因组的工具库。
