利用CRISPR/Cas9系统定向改良水稻稻瘟病抗性
收稿日期: 2019-04-10
修回日期: 2019-05-24
网络出版日期: 2019-07-10
基金资助
中国农业科学院创新工程超级稻育种创新团队和水稻杂种优势研究机理研究创新团队资助项目(CAAS-ASTIP-2013-CNRRI);转基因重大专项(2016ZX08001-002)
Orientation Improvement of Blast Resistance in Rice via CRISPR/Cas9 System
Received date: 2019-04-10
Revised date: 2019-05-24
Online published: 2019-07-10
【目的】CRISPR/Cas9基因编辑技术是作物遗传改良的有效工具。本研究通过对水稻Pita、Pi21和ERF922稻瘟病相关基因进行定点编辑,以期获得能够稳定遗传的抗稻瘟病水稻材料。【方法】利用CRISPR/Cas9基因编辑技术,以Pita、Pi21和ERF922为靶基因,构建共编辑载体pC1300-2×35S::Cas9-gPita-gPi21-gERF922 ,用农杆菌转化长粒粳稻恢复系L1014,筛选获得稳定遗传的纯合突变体用于稻瘟病抗性鉴定。【结果】在T0代转基因株系中,Pita、Pi21和ERF922突变频率分别为75%、85%和65%,突变基因型多为双等位突变。筛选到的不含T-DNA成分的T1代能够稳定遗传给T2代,并从中获得Pi21单突变纯合株系及Pita、Pi21和ERF922的三突变纯合株系。稻瘟病抗性鉴定结果表明,与野生型相比,突变株系的抗性显著提高。同时,接种后纯合突变体株系内水杨酸、茉莉酸和乙烯等信号转导途径相关基因的表达量均上调。据此,我们推测纯合突变株系对稻瘟病的抗性增强可能与其对稻瘟病菌的响应被激活有关。【结论】利用CRISPR/Cas9技术获得了能够稳定遗传和具有较高稻瘟病抗性的纯合突变株系,为水稻稻瘟病抗性改良提供了良好的材料。
徐鹏, 王宏, 涂燃冉, 刘群恩, 吴玮勋, 傅秀民, 曹立勇, 沈希宏 . 利用CRISPR/Cas9系统定向改良水稻稻瘟病抗性[J]. 中国水稻科学, 2019 , 33(4) : 313 -322 . DOI: 10.16819/j.1001-7216.2019.9043
【Objective】 CRISPR/Cas9 gene editing is an effective biotechnology for crop genetic improvement. This study aims to edit Pita, Pi21 and ERF922 genes for obtaining valuable and stable genetic materials with resistance to rice blast. 【Method】The target genes Pita, Pi21 and ERF922 were selected for constructing the pC1300-2×35S::Cas9-gPita-gPi21-gERF922 expression vector by CRISPR/Cas9 gene editing technology, and transformed by Agrobacterium-mediated method into long-grain japonica rice restore lines L1014. Stable genetic mutant lines were selected and their resistance to rice blast was identified. 【Result】 In the T0 transgenic lines, the mutation rates of Pita, Pi21 and ERF922 were 75%, 85% and 65%, respectively, and most of the mutant lines are biallelic mutations. The homozygous mutation lines without T-DNA components within the screened T1 were selected and transferred to T2 stably. Evaluation analysis of resistance to rice blast suggested that the mutant lines showed significantly higher resistance to M. oryzae compared with the wild type. The expression level of defense-related genes involved in signaling pathways of salicylic acid, jasmonic acid and ethylene metabolisms was up-regulated in the mutant lines compared to the wild type after inoculation of the physiological races of M. oryzae. Therefore, we hypothesized that the increased resistance of homozygous mutant lines to rice blast might be related to the activation of the response to M. oryzae. 【Conclusion】The homozygous mutant lines with stable inheritance and high blast resistance were obtained by CRISPR/Cas9 system, which would provide ideal materials for improving blast resistance in rice.
Key words： rice; CRISPR/Cas9; Pita; Pi21; ERF922; blast resistance
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