MISSA Is a Highly Efficient in Vivo DNA Assembly Method for Plant Multiple-Gene Transformation
MISSA Is a Highly Efficient in Vivo DNA Assembly Method for Plant Multiple-Gene Transformation
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DOI：
10.1104/pp.109.152249
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发表时间：
2010-05-01
期刊：
影响因子：
7.4
通讯作者：
Wang, Xue-Chen
中科院分区：
文献类型：
--
作者：
Chen, Qi-Jun;Xie, Min;Wang, Xue-Chen
我们描述了一种高效的体内DNA组装方法，即多轮体内定点组装（MISSA），该方法有利于植物多基因转化。 MISSA 基于由供体菌株驱动的接合转移和两个体内位点特异性重组事件（由受体菌株中的诱导型 Cre 重组酶和噬菌体 lambda 位点特异性重组蛋白介导），以实现多个转基因 DNA 的体内转移和体内组装。通过交替使用两个专门设计的供体载体，可以循环迭代地进行组装反应。作为原理验证实验，我们构建了一些植物多基因二元载体。其中一种载体是通过 15 轮 MISSA 反应生成的，并在转基因拟南芥 (Arabidopsis thaliana) 中得到了证实。由于 MISSA 将繁琐且耗时的体外操作简化为细菌菌株的简单混合，因此将大大节省与多个转基因或合成 DNA 组装相关的时间、精力和费用。 MISSA 的基本原理适用于所有生物体（例如大肠杆菌、酵母、植物和动物）中的多基因性状、生物合成途径或蛋白质复合物的工程设计。 MISSA 在合成生物学方面也有潜在的应用，无论是基础理论还是应用生物技术，旨在组装遗传途径，从天然或合成 DNA 生产生物燃料、药品和工业化合物。
We describe a highly efficient in vivo DNA assembly method, multiple-round in vivo site-specific assembly (MISSA), which facilitates plant multiple-gene transformation. MISSA is based on conjugational transfer, which is driven by donor strains, and two in vivo site-specific recombination events, which are mediated by inducible Cre recombinase and phage lambda site-specific recombination proteins in recipient strains, to enable in vivo transfer and in vivo assembly of multiple transgenic DNA. The assembly reactions can be performed circularly and iteratively through alternate use of the two specially designed donor vectors. As proof-of-principle experiments, we constructed a few plant multigene binary vectors. One of these vectors was generated by 15 rounds of MISSA reactions and was confirmed in transgenic Arabidopsis (Arabidopsis thaliana). As MISSA simplifies the tedious and time-consuming in vitro manipulations to a simple mixing of bacterial strains, it will greatly save time, effort, and expense associated with the assembly of multiple transgenic or synthetic DNA. The principle that underlies MISSA is applicable to engineering polygenic traits, biosynthetic pathways, or protein complexes in all organisms, such as Escherichia coli, yeast, plants, and animals. MISSA also has potential applications in synthetic biology, whether for basic theory or for applied biotechnology, aiming at the assembly of genetic pathways for the production of biofuels, pharmaceuticals, and industrial compounds from natural or synthetic DNA.