Targeted Mutagenesis of the Rice FW 2.2-Like Gene Family Using the CRISPR/Cas9 System Reveals OsFWL4 as a Regulator of Tiller Number and Plant Yield in Rice
Targeted Mutagenesis of the Rice FW 2.2-Like Gene Family Using the CRISPR/Cas9 System Reveals OsFWL4 as a Regulator of Tiller Number and Plant Yield in Rice
复制标题
使用 CRISPR/Cas9 系统对水稻 FW 2.2 样基因家族进行定向诱变，揭示 OsFWL4 作为水稻分蘖数和植物产量的调节因子
DOI：
10.3390/ijms21030809
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发表时间：
2020-02-01
影响因子：
5.6
通讯作者：
Zhao, Xiangxiang
中科院分区：
文献类型：
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作者：
Gao, Qingsong;Li, Gang;Zhao, Xiangxiang
FW2.2-like(FWL)基因编码富含半胱氨酸的蛋白，具有胎盘特异的8结构域。它们在细胞分裂和器官大小控制、对根瘤菌侵染的反应以及植物中金属离子的动态平衡中发挥作用。在这里，我们使用农杆菌介导的CRISPR/Cas9系统来靶向8个水稻FWL基因。我们成功地为16个靶标中的15个产生了转基因T-0系。在所有15个靶点的T0系中均检测到靶向突变，平均突变率为81.6%。转移DNA(T-DNA)截断是T0植株诱变失败的主要原因。T-DNA分离分析表明，在T-1代，转基因植株中的T-DNA插入片段很容易被消除。在检查的30个假定的靶外位点中，有13个位点检测到意外突变。表型分析表明，OsFWL4基因突变体的分蘖数和单株产量显著高于野生型。OsFWL4基因突变体的旗叶宽于野生型。突变体叶片宽度的增加是由细胞数量的增加引起的。此外，OsFWL1基因突变体的籽粒长度高于野生型。我们的结果表明，利用农杆菌介导的CRISPR/Cas9系统，可以在T-1世代获得目标突变的无转基因水稻植株，并且OsFWL4基因是分蘖数和植株产量的负调控基因。
The FW2.2-like (FWL) genes encode cysteine-rich proteins with a placenta-specific 8 domain. They play roles in cell division and organ size control, response to rhizobium infection, and metal ion homeostasis in plants. Here, we target eight rice FWL genes using the CRISPR/Cas9 system delivered by Agrobacterium-mediated transformation. We successfully generate transgenic T-0 lines for 15 of the 16 targets. The targeted mutations are detected in the T0 lines of all 15 targets and the average mutation rate is found to be 81.6%. Transfer DNA (T-DNA) truncation is a major reason for the failure of mutagenesis in T0 plants. T-DNA segregation analysis reveals that the T-DNA inserts in transgenic plants can be easily eliminated in the T-1 generation. Of the 30 putative off-target sites examined, unintended mutations are detected in 13 sites. Phenotypic analysis reveals that tiller number and plant yield of OsFWL4 gene mutants are significantly greater than those of the wild type. Flag leaves of OsFWL4 gene mutants are wider than those of the wild type. The increase in leaf width of the mutants is caused by an increase in cell number. Additionally, grain length of OsFWL1 gene mutants is higher than that of the wild type. Our results suggest that transgene-free rice plants with targeted mutations can be produced in the T-1 generation using the Agrobacterium-mediated CRISPR/Cas9 system and that the OsFWL4 gene is a negative regulator of tiller number and plant yield.