PcMuORP1, an OxathiapiprolinResistance Gene, Functions as a Novel Selection Marker for Phytophthora Transformation and CRISPR/Cas9 Mediated Genome Editing
PcMuORP1, an OxathiapiprolinResistance Gene, Functions as a Novel Selection Marker for Phytophthora Transformation and CRISPR/Cas9 Mediated Genome Editing
复制标题
PcMuORP1 是一种 oxathiapiprolin 抗性基因，可作为疫霉菌转化和 CRISPR/Cas9 介导的基因组编辑的新型选择标记
DOI：
10.3389/fmicb.2019.02402
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发表时间：
2019-10-22
影响因子：
5.2
通讯作者：
Liu, Xili
中科院分区：
文献类型：
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作者：
Wang, Weizhen;Xue, Zhaolin;Liu, Xili
疫霉属（Phytophthora）是卵菌纲的一个属，含有许多毁灭性的植物病原体，在世界范围内造成巨大的经济损失。最近，基于CRISPR/Cas9的基因组编辑工具被引入疫霉属，以描述单个基因的功能。然而，可用于疫霉属植物转化的选择标记有限，这在某些情况下会限制转基因操作。我们假设，PcMuORP1，一种来自辣椒疫霉的内源性杀真菌剂抗性基因，通过ORP1蛋白中改变的靶位点赋予对杀真菌剂oxathiapiprolin的抗性，可以用作替代标记。为了验证这一假设，将基因PcMuORP1引入CRISPR/Cas9系统，并使用其作为选择标记实现了辣椒疫霉中缺失基因的互补。所有抗氧硫匹灵的转化子均被证实含有标记基因，表明阳性筛选率为100%。该标记也可用于大豆疫霉、荔枝疫霉等代表性疫霉种的筛选，阳性筛选率均为100%。此外，比较研究表明，与常规选择标记NPT II相比，使用PcMuORP1导致高得多的筛选效率，特别是在辣椒疫霉中。在没有选择压力的情况下连续传代和无性繁殖会导致转化体中选择标记的丢失，这表明PcMuORP1基因对转化体的适应性没有长期影响，可以在后续项目中重复使用作为选择标记。因此，我们已经创建了一个替代的选择标记，通过使用杀真菌剂抗性基因，这将加快在这些物种中的基因的功能研究疫霉转化。
Phytophthora, a genus of oomycetes, contains many devastating plant pathogens, which cause substantial economic losses worldwide. Recently, CRISPR/Cas9-based genome editing tool was introduced into Phytophthora to delineate the functionality of individual genes. The available selection markers for Phytophthora transformation, however, are limited, which can restrain transgenic manipulation in some cases. We hypothesized that PcMuORP1, an endogenous fungicide resistance gene from P. capsici that confers resistance to the fungicide oxathiapiprolin via an altered target site in the ORP1 protein, could be used as an alternative marker. To test this hypothesis, the gene PcMuORP1 was introduced into the CRISPR/Cas9 system and complementation of a deleted gene in P. capsici was achieved using it as a selection marker. All of the oxathiapiprolin-resistant transformants were confirmed to contain the marker gene, indicating that the positive screening rate was 100%. The novel selection marker could also be used in other representative Phytophthora species including P. sojae and P. litchii, also with 100% positive screening rate. Furthermore, comparative studies indicated that use of PcMuORP1 resulted in a much higher efficiency of screening compared to the conventional selection marker NPT II, especially in P. capsici. Successive subculture and asexual reproduction in the absence of selective pressure were found to result in the loss of the selection marker from the transformants, which indicates that the PcMuORP1 gene would have little long term influence on the fitness of transformants and could be reused as the selection marker in subsequent projects. Thus, we have created an alternative selection marker for Phytophthora transformation by using a fungicide resistance gene, which would accelerate functional studies of genes in these species.