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Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase

机译:Bxb1重组酶介导的柳枝poll花粉转基因自切除

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Background Switchgrass ( Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated. Results A novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/ att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used. Conclusion The results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches.
机译:背景柳枝Pan(Panicum virgatum L.)作为生产生物基塑料,化学品和能源的平台具有巨大潜力,这主要是由于其在边缘土地上的生物量高且农业投入低。在过去的十年中,通过转基因方法对该作物进行遗传改良的兴趣日益增加。由于像大多数多年生禾本科草一样,柳枝exclusively完全是异花授粉且驯养差,因此,防止这种转基因花粉散布到环境中是这种重要生物量作物商业化应用的关键条件。在这项研究中,研究了使用大的丝氨酸位点特异性重组酶Bxb1控制花粉介导的转基因柳枝gene中的基因流的可行性。结果使用一种新颖的方法,利用两个单独载体的共转化来测试柳枝the中Bxb1 / att重组系统的功能。另外,鉴定了两个具有高花粉特异性活性的启动子,并在将其引入显式设计用于重组的自动切割和定量分析的测试载体之前对其进行了全面表征。我们对柳枝poll花粉中重组酶和标记基因进行精确编程切除的开发策略导致产生了转基因切除的后代。自动转化效率在22-42%的范围内,具体取决于转化事件和所用的分析方法。结论此处提出的结果标志着建立可靠的柳枝bio生物控制系统的重要里程碑,该系统将通过先进的生物技术方法促进该作物作为生物精炼原料的发展。

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