基于CRISPR/Cas9技术的水稻pi21基因编辑材料的创制及稻瘟病抗性鉴定

Generation of Rice pi21 Gene Editing Lines Based on CRISPR/Cas9 Technology and Evaluation of Their Blast Resistance

利用CRISPR-Cas9技术,对水稻稻瘟病感病品种日本晴pi21基因进行定点突变,获得了抗稻瘟病的pi21隐性突变株系。基因编辑载体T0转化植株的检测结果表明,86.7%的T0植株的pi21基因靶序列发生了碱基缺失。通过T-DNA序列的PCR检测,剔除携带T-DNA的T1植株,从23个T1株系筛选获得107个不含T-DNA转基因序列的植株。然后对pi21的编辑位点进行酶切和测序分析,获得了21株pi21纯合突变体。对1个pi21突变株系进行稻瘟病菌孢子喷雾接种,与未转化日本晴相比,突变株系显著抗病。对接种水稻样品的6个病程相关基因进行q RT-PCR分析,与感病日本晴比较,pi21突变株系在接种稻瘟病菌后,病程相关基因诱导表达的速度更快,表达量更高。本研究利用基因编辑技术,实现了对感稻瘟病水稻的定点突变并提高了其稻瘟病抗性水平,为利用该技术培育持久抗稻瘟病水稻品种奠定了研究基础。

pi21 gene was targeted editing using CRISPR/Cas9 technology in the blast susceptible rice variety Nipponbare and a set of pi21 recessive mutants exhibiting enhanced rice blast resistance were obtained. The results showed that 86.7% of T0 transgenic plants had nucleotide deletions of pi21 target sequence. Through T-DNA specific PCR, the T1 generation plants containing T-DNA were eliminated, and 107 T-DNA-free plants were identified from 23 T1 transgenic lines. Restriction digestion and sequencing analysis revealed that 21 out of 107 rice plants were pi21 homozygous mutants. One pi21 mutant line was inoculated with M. oryzae, showing significantly enhanced rice blast resistance compared with the untransformed Nipponbare. q RT-PCR analysis showed that the expression of the tested pathogenesis-related genes were more rapidly and strongly induced in the pi21 mutant plants compared to the blast susceptible Nipponbare during fungal infection. In this study, the targeted editing of pi21 gene in the blast susceptible variety and improved resistance to rice blast was achieved, which provides the foundation for breeding durable rice blast resistant rice varieties.