| 林春姿，黄其伟，侯 艳，高 上，韩志开，韦淡虹，陈 淳，王加峰.水稻多胺氧化酶基因（OsPAO4）CRISPR/Cas9 编辑突变体的创制[J].广东农业科学,2023,50(3):1-10 |
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水稻多胺氧化酶基因（OsPAO4）CRISPR/Cas9 编辑突变体的创制 |
| Construction of Rice Mutants of the Polyamine Oxidase Gene OsPAO4 Based on CRISPR/Cas9 Editing |
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| DOI：10.16768/j.issn.1004-874X.2023.03.001 |
| 中文关键词: 水稻 OsPAO4 CRISPR/Cas9 基因编辑 多胺氧化酶 |
| 英文关键词: rice OsPAO4 CRISPR/Cas9 gene editing polyamine oxidase |
| 基金项目:广东省自然科学基金项目（2019A1515011825）；广东省水稻育种新技术重点实验室 2020 年开放课题 |
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| 中文摘要: |
| 【目的】稻瘟病是水稻生产上的重要限制因素，挖掘与利用抗病基因是实现水稻高产稳产的重要保障。前期研究发现，稻瘟病抗性相关 osa-miR-21 可能通过靶向调控多胺氧化酶基因 OsPAO4 调节水稻稻瘟病抗性，但目前多胺氧化酶（Polyamine oxidases，PAOs）在水稻抗病中的功能研究尚未见报道。为进一步探究其在水稻抗病中的可能生物学功能，本研究利用 CRISPR/Cas9 编辑技术对水稻 OsPAO4 基因进行定点编辑并对其后代突变体进行分析。【方法】在 OsPAO4 第 2 外显子处设计了 1 个 20 bp 的编辑靶点，将靶点核苷酸片段克隆至 pRGEB32 载体，获得 OsPAO4 编辑载体。随后，利用农杆菌介导法转化水稻 Pik-H4 NIL 愈伤组织中，经过再生培养、潮霉素检测获得转基因阳性植株，并对 T0 代植株靶位点附近 DNA 序列进行 PCR 和测序分析。【结果】OsPAO4 基因被成功编辑，最终获得 25 个转基因阳性植株，并在 T0 代产生多种突变类型：3 株纯合突变、18 株杂合突变和 4 株未发生编辑的植株。此外，T0 代杂合突变 ospao4-8 的颖壳颜色由紫色变为深灰色，且ospao4 突变体中过氧化氢酶（Catalase，CAT）活性有所升高，推测 OsPAO4 可能参与水稻的基础免疫过程。【结论】创制了多种类型 ospao4 突变体材料，初步确定 OsPAO4 可能参与水稻免疫过程的调控，为进一步深入揭示OsPAO4 基因的具体生物学功能和分子调控机制奠定了重要基础。 |
| 英文摘要: |
| 【Objective】Rice blast is an important limiting factor in rice production. Exploring and utilizing resistant genes is an important guarantee for achieving high and stable yield of rice. Previous studies have shown that rice blast resistance-related microRNA osa-miR-21 may regulate rice blast resistance by targeting the polyamine oxidase gene OsPAO4. However, the function of Polyamine Oxidases (PAOs) in rice disease resistance has not been reported. In order to further explore the possible biological function of OsPAO4 gene in rice disease resistance, the CRISPR/Cas9 editing technology was used for site-specific editing of ospao4 gene mutants and the edited offspring were also analyzed.【Method】A 20-bp editing target was designed in the second exon of OsPAO4, and the nucleotide fragments of the target was cloned into pRGEB32 vector to obtain OsPAO4 editing vector. Then, the rice Pik-H4 NIL callus was transformed by Agrobacteriummediated method. Positive transgenic plants were obtained by regeneration culture and hygromycin detection, PCR and sequence analysis were performed on the DNA sequences near the target site in the T0 generation plants.【Result】The results show that the OsPAO4 gene was successfully edited and finally 25 transgenic-positive plants were obtained, and various mutation types were produced in the T0 generation, including 3 homozygous mutants, 18 heterozygous mutants and 4 nonedited plants. In addition, the glume color of the heterozygous mutation ospao4-8 of the T0 generation changed from purple to dark gray, and the activity of catalase (CAT) in the mutants was increased. It was predicted that OsPAO4 might participate in the basis immunization process.【Conclusion】A variety of ospao4 mutants were created in this study, and it was preliminarily determined that OsPAO4 might participate in the regulation of rice immune process, which laid an important foundation for further revealing the specific biological function and molecular regulation mechanism of OsPAO4 gene. |
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