| 林春姿，谢华斌，黄其伟，高 上，王加峰.利用 CRISPR/Cas9 技术定向编辑水稻 OsIQD1 基因[J].广东农业科学,2022,49(8):1-10 |
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利用 CRISPR/Cas9 技术定向编辑水稻 OsIQD1 基因 |
| Targeted Editing of OsIDQ1 Gene in Rice Based on CRISPR /Cas9 Technology |
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| DOI：10.16768/j.issn.1004-874X.2022.08.001 |
| 中文关键词: 水稻 钙调素结合蛋白 OsIQD1 CRISPR/Cas9 基因编辑 |
| 英文关键词: rice calmodulin binding protein OsIQD1 CRISPR/Cas9 gene editing |
| 基金项目:广东省自然科学基金（2019A1515011825） |
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| 中文摘要: |
| 【目的】植物特异性 IQD 家族蛋白质是一类植物特有的钙调素结合靶蛋白，在植物的生物防
御和发育调节中发挥重要作用。前期酵母双杂筛选试验表明，Pik-H4 与 OsIQD1 存在互作关系，为进一步
揭示 OsIQD1 在水稻抗稻瘟病中的生物学功能，利用 CRISPR/Cas9 编辑技术对 OsIQD1 基因进行定点编辑。
【方法】通过序列比对获得水稻 OsIQD1。在 OsIQD1 第 1 外显子和第 5 外显子（DUF4005 结构域）区域设计 2
个 20 bp 的编辑靶点，将 2 个靶点核苷酸片段克隆至 pRGEB32 载体，获得 pRGEB32-OsIQD1-gRNA 载体，利用
农杆菌介导法转化水稻 Pik-H4 NIL 愈伤组织，经再生培养、潮霉素检测获得转基因阳性植株，并对 T0 代转基
因植株靶点区域序列进行 PCR 和测序，分析 osiqd1 的突变类型。【结果】转基因再生植株经过对抗潮霉素基因
HPT Ⅱ的 PCR 鉴定，获得 30 株阳性株系。对 T0 代植株靶点附近序列的测序结果表明，OsIQD1 基因被成功编
辑，两个位点的编辑效率分别为 21.67% 和 26.67%，其中，靶点 2 区域有 1 个纯合突变株系。【结论】研究结
果为进一步利用 osiqd1 突变体开展其参与钙离子 / 钙调素信号转导通路的机制研究提供了遗传材料，初步推定
OsIQD1 参与植物的基础免疫反应。 |
| 英文摘要: |
| 【Objective】IQD gene family proteins are plant specific calmodulin-binding target proteins, which play an
important role in plant defense and development regulation. The interaction between Pik-H4 and OsIQD1 was found in the
yeast double hybrid screening experiment in laboratory. In order to reveal the specific biological functions of OsIQD1 in rice
blast resistance, OsIQD1 gene was edited by using CRISPR/Cas9 editing technique.【Method】OsIQD1 was obtained by
sequence alignment. Two 20 bp editing targets were designed in exon 1 and exon 5 of OsIQD1 (DUF4005 domain), and the
nucleotide fragments of the two targets were cloned into pRGEB32 vector to obtain pRGEB32-OsIQD1-gRNA vector. The
rice Pik-H4 NIL callus was transformed by agrobacterium-mediated method. Transgenic positive plants were obtained by
regeneration culture and hygromycin resistance screening. PCR and sequencing were performed on the target region sequences of transgenic plants of T0
generation to analyze the mutation types of osiqd1.【Result】The transgenic regenerated plants
were identified by PCR with hygromycin resistance gene HPT Ⅱ , and 30 positive lines were obtained. Sequencing analysis
of the sequences near the target site of T0
generation plants showed that OsIQD1 gene was successfully edited, and the editing
efficiency of the two sites was 21.67% and 26.67%, respectively. There was one homozygous mutant line in target 2 region.
【Conclusion】The study results provide genetic material for further studies on the mechanism of osiqd1 mutation involved in
calcium ion/calmodulin signaling pathway, and OsIQD1 is preliminarily presumed to be involved in the basic immune response
of plants. |
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