Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion.
Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion.
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DOI：
10.1186/s12896-015-0212-2
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发表时间：
2015-10-09
影响因子：
3.5
通讯作者：
Srivastava V
中科院分区：
文献类型：
--
作者：
Nandy S;Zhao S;Pathak BP;Manoharan M;Srivastava V
多基因转化和基因叠加的实用方法对于在转基因作物中工程化复杂性状和添加新性状是极其重要的。通过基因堆叠进行性状配置将大大简化下游植物育种和性状渐渗到栽培品种中。基因堆积到预定的基因组位点取决于靶向DNA整合和选择标记基因再循环的机制。靶向整合到由核酸酶产生的染色体断裂中需要大的转化努力。另一方面，诸如Cre-lox的酶有效地驱动植物中的位点特异性整合。然而，Cre-lox重组的可逆性，由于纳入两个顺式定位的lox位点，提出了一个主要的瓶颈，在其应用中的基因堆叠。在这里，我们描述了一种策略，解决这个瓶颈，通过切除一个顺式定位的脂氧合酶，嵌入在标记基因，由核酸酶活性。所有的转基因株系都是通过用质粒构建体对水稻愈伤组织进行粒子轰击而获得的。使用标准分子方法构建构建体。通过PCR、Southern杂交和DNA测序分析转基因位点。我们开发了一种高效的基因堆叠方法，利用强大的重组酶，如Cre-lox和FLP-FRT，位点特异性基因整合，和核酸酶标记基因切除。我们在水稻中产生Cre介导的位点特异性整合位点，并显示I-SceI以约20%的效率切除标记基因，无缝连接位点中的基因。接下来，我们展示了ZFN可以用于标记切除，并且该基因座可以通过重组酶再次靶向。因此，我们将重组酶的能力扩展到植物中的基因堆叠应用。最后，我们表明，热诱导I-SceI也适合标记切除，因此可以作为一个重要的工具，简化这个基因堆叠平台。开发了一种在植物细胞中进行基因堆叠的实用方法，该方法允许通过多轮转化插入靶向基因，这是将新性状引入转基因品系以在田间快速部署所需的方法。通过使用Cre-lox，一种强大的位点特异性重组系统，该方法大大提高了基因堆叠效率，并通过核酸酶的应用，在预定的染色体位点上开发了无标记的无缝基因堆叠。
Practical approaches for multigene transformation and gene stacking are extremely important for engineering complex traits and adding new traits in transgenic crops. Trait deployment by gene stacking would greatly simplify downstream plant breeding and trait introgression into cultivars. Gene stacking into pre-determined genomic sites depends on mechanisms of targeted DNA integration and recycling of selectable marker genes. Targeted integrations into chromosomal breaks, created by nucleases, require large transformation efforts. Recombinases such as Cre-lox, on the other hand, efficiently drive site-specific integrations in plants. However, the reversibility of Cre-lox recombination, due to the incorporation of two cis-positioned lox sites, presents a major bottleneck in its application in gene stacking. Here, we describe a strategy of resolving this bottleneck through excision of one of the cis-positioned lox, embedded in the marker gene, by nuclease activity. All transgenic lines were developed by particle bombardment of rice callus with plasmid constructs. Standard molecular approach was used for building the constructs. Transgene loci were analyzed by PCR, Southern hybridization, and DNA sequencing. We developed a highly efficient gene stacking method by utilizing powerful recombinases such as Cre-lox and FLP-FRT, for site-specific gene integrations, and nucleases for marker gene excisions. We generated Cre-mediated site-specific integration locus in rice and showed excision of marker gene by I-SceI at ~20 % efficiency, seamlessly connecting genes in the locus. Next, we showed ZFN could be used for marker excision, and the locus can be targeted again by recombinases. Hence, we extended the power of recombinases to gene stacking application in plants. Finally, we show that heat-inducible I-SceI is also suitable for marker excision, and therefore could serve as an important tool in streamlining this gene stacking platform. A practical approach for gene stacking in plant cell was developed that allows targeted gene insertions through rounds of transformation, a method needed for introducing new traits into transgenic lines for their rapid deployment in the field. By using Cre-lox, a powerful site-specific recombination system, this method greatly improves gene stacking efficiency, and through the application of nucleases develops marker-free, seamless stack of genes at pre-determined chromosomal sites.