High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate
High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate
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DOI：
10.1104/pp.110.169581
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发表时间：
2011-04-01
期刊：
影响因子：
7.4
通讯作者：
Snell, Kristi D.
中科院分区：
文献类型：
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作者：
Bohmert-Tatarev, Karen;McAvoy, Susan;Snell, Kristi D.
利用操纵子延伸策略设计了用于烟草（Nicotiana tabacum）质体转化以生产可再生的、可生物降解的塑料聚羟基丁酸酯（PHB）的优化的遗传构建体。基于编码PHB途径酶的细菌基因与烟草质体组的密码子使用和GC含量的相似性，选择它们用于该构建体。与宿主质体具有有限同源性但已知产生高水平质体重组蛋白的调控元件用于增强转基因的表达。含有PHB途径的基因和编码大观霉素抗性的选择标记基因的部分转录单位在5'端侧接宿主植物的psbA编码序列，在3'端侧接宿主植物的3' psbA非翻译区。这种设计允许将转基因插入质体中作为psbA操纵子的延伸，使得不需要添加启动子来驱动转基因的表达。将优化的构建体转化到烟草中并随后对转基因植物进行壮观霉素选择，产生了能够在叶组织样品中产生高达18.8%干重的PHB的T0植物。这些植物是可育的，并产生有活力的种子。还分离出在叶组织样品中产生高达17.3%干重的PHB和在植物的总生物量中产生高达8.8%干重的PHB的T1植物。
An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5' end by the host plant's psbA coding sequence and at the 3' end by the host plant's 3' psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.