中国水稻科学 ›› 2022, Vol. 36 ›› Issue (3): 248-258.DOI: 10.16819/j.1001-7216.2022.211007

• 研究报告 • 上一篇    下一篇

利用CRISPR/Cas9技术创制抗稻瘟病香型早籼温敏核不育系

梁敏敏#, 张华丽#, 陈俊宇, 戴冬青, 杜成兴, 王惠梅, 马良勇*()   

  1. 中国水稻研究所 水稻生物学国家重点实验室/国家水稻改良中心,杭州 310006
  • 收稿日期:2021-10-25 修回日期:2021-12-20 出版日期:2022-05-10 发布日期:2022-05-11
  • 通讯作者: 马良勇
  • 作者简介:第一联系人:

    #共同第一作者

  • 基金资助:
    中央级公益性科研院所基本科研业务费专项(CPSIBRF-CNRRI-202105);水稻生物学国家重点实验室开放课题(20210207);水稻生物学国家重点实验室开放课题(20190110);中国农业科学院科技创新工程资助项目。

Developing Fragrant Early indica TGMS Line with Blast Resistance by Using CRISPR/Cas9 Technology

LIANG Minmin#, ZHANG Huali#, CHEN Junyu, DAI Dongqing, DU Chengxing, WANG Huimei, MA Liangyong*()   

  1. State Key Laboratory of Rice Biology and Chinese National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 310006, China
  • Received:2021-10-25 Revised:2021-12-20 Online:2022-05-10 Published:2022-05-11
  • Contact: MA Liangyong
  • About author:First author contact:

    #These authors contributed equally to this work

摘要:

【目的】创制新型抗稻瘟病香型早籼温敏核不育系,为高产优质杂交水稻选育提供资源。【方法】利用CRISPP/Cas9技术在水稻稻瘟病基因Pi21、温敏不育基因TMS5和香味基因Badh2的第1外显子处设计靶位点,构建多基因表达载体pC1300-2×35S::gTMS5-gBadh2-gPi21,转化优质常规籼稻品种中早70,测序鉴定分析获得纯合阳性稳定株系。利用稻瘟病喷雾接种和打孔接种方法对稻瘟病基因Pi21的纯合突变株系进行稻瘟病抗性鉴定,利用GC-MS技术对Badh2纯合突变株系的香味物质2-乙酰-1-吡咯啉(2-AP)含量进行测定。【结果】在T0转基因株系中,Pi21TMS5Badh2突变频率分别为87.5%、80.0%和87.5%,突变类型多为双等位突变。从T1代中筛选不含载体骨架的纯合突变株系,获得两种三突变纯合株系。稻瘟病接种结果表明,与野生型相比,T2Pi21纯合变异株系的抗性显著提高。同时,接种后纯合突变体株系内相关防卫基因的表达量显著上调,ROS积累量也显著增加。tms5纯合变异株系表现出典型的温敏不育特性,TMS5基因的表达水平与野生型相比显著降低,高温下UbL404基因的表达水平明显高于野生型。与野生型相比,在Badh2纯合突变体植株中Badh2的表达水平显著下调,并且香味物质2-AP含量极显著增加。【结论】利用CRISPR/Cas9技术成功对Pi21、TMS5Badh2基因同时进行定向编辑,获得了具有高抗稻瘟病的香型温敏不育系,为高抗、香型不育系材料的选育提供参考,加快高产优质杂交水稻的选育。

关键词: 水稻, CRISPR/Cas9, Badh2, Pi21, TMS5

Abstract:

【Objective】 New thermosensitive genic male sterile lines with high resistance and fragrance quality will provide resources for hybrid rice breeding. 【Method】The target genes Pi21, TMS5 and Badh2 were selected to construct the pC1300-2×35S::gTMS5-gBadh2-gPi21expression vector by CRISPR/Cas9 technology for transformation of early indica rice Zhongzao 70. The positive transgenic plants were obtained through sequencing analysis. Magnaporthe oryzae spraying inoculation and punching inoculation assays were conducted to identify the resistance to rice blast, and the content of 2-acetyl-1-pyrroline was measured by GC-MS. 【Result】In T0 transgenic lines, the mutation rates of Pi21, TMS5 and Badh2 were 87.5%, 80.0% and 87.5%, respectively, and most of the mutant lines are biallelic mutations. Two types of the homozygous triple mutants without the vector were obtained in T1 transgenic lines. Evaluation analysis of resistance to rice blast suggested that T2 generation of the pi21 homozygous mutant lines showed significantly higher resistance to M. oryzae compared with the wild type. The expression levels of defense-related genes were up-regulated and the ROS accumulation increased in the homozygous mutant lines compared to the wild type after inoculation of M. oryzae. tms5 homozygous mutant lines displayed thermo-sensitive male sterility. Compared with the wild type, the expression level of TMS5 was significantly decreased and the transcription of UbL404 was increased in the tms5 homozygous mutants at high temperature. The expression level of Badh2 was down-regulated and the content of 2-AP increased dramatically in the Badh2 homozygous mutant lines. 【Conclusion】The homozygous triple mutant lines of Pi21, TMS5 and Badh2 were obtained by CRISPR/Cas9-mediated technology, which would provide materials for high-resistant and fragrant rice breeding.

Key words: rice, CRISPR/Cas9, Badh2, Pi21, TMS5