We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5′‐untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High‐level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.

中文翻译：

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ORF在双顺反子操纵子中的独立翻译，这是多顺反子叶绿体基因表达的合成构件。

我们设计了一个双顺反子质体标记系统，该系统依赖于该质体翻译多顺反子mRNA的能力。转质体克隆的鉴定基于对第一个开放阅读框（ORF）中编码的抗生素抗性的选择以及第二个ORF中编码的烟草叶绿体中报告基因产物的积累。基于aadA或neo的表达，抗生素抗性基因可以编码壮观霉素或卡那霉素抗性基因分别。该研究中使用的报告基因是绿色荧光蛋白（GFP）。mRNA水平取决于第一个ORF的5'-非翻译区。蛋白质输出取决于核糖体结合的强度，并与可翻译的mRNA水平成正比。由于未处理双顺反子mRNA，因此我们可以证明第二个ORF的蛋白质输出独立于第一个ORF。来自第二个ORF的高水平GFP积累有助于鉴定紫外线下的转质体事件。从未加工的mRNA表达多种蛋白质是一项实验设计，可实现多顺反子mRNA的可预测蛋白质输出，从而扩展了植物合成生物学的工具包。