Expression of bar in the Plastid Genome Confers Herbicide Resistance

Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT. We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (>7% of total soluble cellular protein). Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use. We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen.

草丁膦（Phosphinothricin, PPT）是一类环境友好型非选择性除草剂的活性成分。此前已有研究报道，通过核表达编码草丁膦乙酰转移酶（phosphinothricin acetyltransferase）的bar转基因，可使转基因作物获得对PPT的抗性。本研究报道了在烟草（Nicotiana tabacum cv Petit Havana）质体中表达细菌bar基因（b-bar1）的研究结果，该基因可使植株获得对含PPT的除草剂Liberty的田间级抗性。本研究还描述了第二个细菌bar基因（b-bar2），以及经密码子优化的合成bar基因（s-bar），这类基因在质体中的表达量显著提升（可达细胞可溶性总蛋白的7%以上）。尽管上述基因的表达水平较高，但目前通过直接筛选尚未获得质体转基因克隆，这表明在直接筛选转基因克隆时，亚细胞定位而非酶的绝对表达量是关键影响因素。经密码子修饰的s-bar基因在常见肠道细菌大肠杆菌（Escherichia coli）中表达量极低，这源于二者密码子使用偏好性的差异。我们提出可利用密码子使用偏好性的差异作为预防措施，以避免在极罕见的质体至细菌的水平基因转移事件中标记基因发生表达。将bar基因定位于质体基因组是替代核基因组整合的极具吸引力的方案，因为质体编码的基因不会通过花粉进行传递。