Klaus Sebastian M J, Huang Fong-Chin, Golds Timothy J, Koop Hans-Ulrich
Nat Biotechnol. 2004 Feb;22(2):225-9. doi: 10.1038/nbt933. Epub 2004 Jan 18.
Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops, the possibility of marker transfer to wild relatives or microorganisms cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.
高等植物质体的基因工程通常涉及稳定导入抗生素抗性基因作为选择标记。尽管在大多数作物中叶绿体基因是母系遗传的，但标记基因转移到野生近缘种或微生物的可能性无法完全排除。此外，标记基因的表达可能会造成大量的代谢消耗。因此，需要有效的方法从质体转化体中完全去除标记基因。从高等植物质体中去除选择基因的一种方法基于环出重组，这一过程难以控制，因为同质性转化体的选择是不可预测的。另一种方法使用CRE/lox系统，但需要额外的再转化和有性杂交来导入和随后去除CRE重组酶。在这里，我们描述了利用野生型色素沉着的重建与质体转化载体相结合，在烟草中产生无标记叶绿体转化体的方法，该载体可防止卡那霉素选择标记的稳定整合。使用突变体的方法的一个优点是，无标记质体转化体可以在第一代（T0）直接产生，而无需再转化或杂交。
