| 冯彦钊，朱庆锋，薛 皦，陈 沛，于 洋.利用 CRIS PR/Cas9 技术创制水稻中花 11 号
dwarf1 突变体[J].广东农业科学,2022,49(11):110-118 |
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利用 CRIS PR/Cas9 技术创制水稻中花 11 号
dwarf1 突变体 |
| Construction of Dwarf1 Mutants in Zhonghua 11 (Oryza sativa L.) by CRISPR/Cas9 |
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| DOI：10.16768/j.issn.1004-874X.2022.11.012 |
| 中文关键词: Gα 亚基 水稻 中花 11 号 突变体 CRISPR-Cas9 |
| 英文关键词: Gα subunit rice Zhonghua 11 mutant CRISPR-Cas9 |
| 基金项目:广东省农业科学院新兴团队项目（202131TD）；广东省农业科学院科技人才引进专项（R2021YJ-QG005，
R2021YJ-YB3011）；广东省自然科学基金（2021A1515110103） |
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| 中文摘要: |
| 【目的】水稻异源三聚体 G 蛋白 α 亚基在植物生长与逆境应答中具有重要的生物学功能，在
水 稻中花 11 号品种中创制 Gα 蛋白编码基因 DWARF1（D1）的突变体，为水稻功能基因组学研究提供材料。
【方法】在中花 11 号的 D1 基因座位上设计靶向其外显子的 sgRNA 序列，构建 CRISPR/Cas9 载体，转化中花 11
号胚愈伤组织，经过抗性愈伤组织筛选和诱导分化后，对再生植株进行鉴定。【结果】筛选到 3 个含有不同编
辑形式的 d1 突变株系，实时荧光定量 PCR 分析发现，D1 mRNA 水平显著降低，表明无义突变介导的 D1 mRNA
降解过程亦被触发。在 3 个 d1 突变株中进一步鉴定到不含潮霉素抗性基因、不含载体骨架的植株，繁育后可以
得到无转基因痕迹、稳定遗传的 d1 敲除植株。对 d1 突变株的生长、发育情况进行观察发现，中花 11 号背景下
d1 突变株显著变矮、穗明显变小，株高和穗长为中花 11 号野生型的 50%~60%。种子变小及变圆，同时，种子
长宽比和千粒重降低约 40%。【结论】获得有效移码突变的中花 11 号背景 d1 突变株系，为进一步研究 G 蛋白
及其相关基因的功能创制遗传材料。 |
| 英文摘要: |
| 【Objective】Heterotrimeric G protein α subunit in rice (Oryza sativa L.) plays important biological roles in
plant growth and stress response. Null mutants of Gα protein encoding gene DWARF1 (D1) in Zhonghua 11 (japonica cv.) are
created, with a view to providing materials for the studies of functional genomics in rice. 【Method】sgRNA was designed
to target the exon at D1 locus in Zhonghua 11, and CRISPR/Cas9 vector was constructed to transform the embryonic calli of
Zhonghua 11. After selection by adding hygromycin, the calli were induced to differentiate and the regenerated plants were
identified.【Result】Three mutant lines harbouring different D1 alleles were identified. Real-time quantitative PCR showed a remarkable decrease in D1 mRNA level, suggesting that nonsense-mediated mRNA decay was also triggered. Progenies
without hygromycin resistance or vector backbone were further identified, representing transgene-free and stably inherited d1
mutants. According to the observation results of the growth and development of d1 mutants, d1 mutants in Zhonghua 11 showed
significantly shorter plant height and smaller panicles. The plant height and panicle length were only 50%-60% compared with
those of the wild-type Zhonghua 11. Smaller and rounder seeds were also shown in d1 mutants, meanwhile, the length/width
ratio and 1000-grain weight were decreased by about 40%.【Conclusion】Effective frame-shift d1 mutants in Zhonghua11 are
obtained, Which creates the genetic materials for functional studies of G protein and other related genes in the future. |
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