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毛兴学, 柳武革, 郑晓钰, 范芝兰, 陈文丰, 潘大建, 李 晨, 王 丰
穗发芽对籼稻生产危害严重。编辑休眠调控基因,创制高休眠性新种质,进而提高穗发芽抗性,是探索改良穗发芽抗性的新途径。泰丰B(TB)是籼型优质杂交稻保持系,主要缺点是种子休眠性偏低,易受穗发芽危害。MPK7/14具有抑制粳稻种子休眠作用,尚不清楚是否适用于籼稻品种。本研究利用 CRISPR/Cas9 技术编辑TB 的MPK7和MPK14基因,通过测序筛选获得纯合突变株系,并利用发芽实验分析基因编辑种子的休眠特性。共获得6个转基因株系,从其后代中筛到2个纯合移码突变株系。纯合突变后代种子休眠性大幅提高,杂合突变后代的休眠性也有明显改善。与TB相比,突变纯合后代株高、分蘖数、穗粒数差异不显著。综上所述,同时敲除MPK7和MPK14可以提高TB种子休眠性,抑制穗发芽和解决水稻穗发芽问题。这些结果为进一步解析穗发芽的抗性机制以及探索新的育种改良方法提供了参考。
关键词:  水稻( Oryza sativa L.)  穗发芽  种子休眠性  CRISPR/Cas9
Generating Pre-harvest Sprouting Resistant Germplasms by Editing MPK7 and MPK14 via CRISPR/Cas9 Technology
MAO Xing-xue, LIU Wu-ge, ZHENG Xiao-yu, FAN Zhi-lan, CHEN Wen-feng, PAN Da-jian, LI Chen, WANG Feng
(Rice Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Key Laboratory of New Technology in Rice Breeding)
Pre-harvest sprouting (PHS) is one of the most serious problems in indica rice production. Generating new germplasm with elevated PHS resistance is applicable by editing dormancy-related gene. Taifeng B (TB) is a maintainer line of indica hybrid rice showing high eating quality, whereas this line shows higher PHS. Two genes MPK7 and MPK14 negatively regulates seed dormancy in japonica rice, but whether both result in elevated PHS resistance in indica rice remains unclear. In this study, in order to generate the germplasms with PHS resistance, we deployed CRISPR/Cas9 technology to edit MPK7 and MPK14 in TB via Agrobacterium-guided transformation. Six lines harboring modified target sequences were screened from the transgenic offspring by sequencing, and two were selected for analyzing the seed dormancy. The results showed that the homozygous mutant significantly delayed the seed germination, rather than heterozygous mutants which also exhibited improved PHS resistance if compared to the wild type TB. No significant difference in plant height, number of tillers and grains per spike between the homozygous mutant and TB was observed. As a result, this study generated PHS resistant lines by CRISPR/Cas9 strategy, which provided theoretical technology and material support for the PHS resistance research and rice breeding.
Key words:  rice (Oryza sativa L.)  pre-harvest sprouting  seed dormancy  CRISPR/Cas9