敲除Bsr-d1和Pi21基因改良优良食味粳稻南粳9108的稻瘟病抗性研究

Improve the blast resistance of Nanjing 9108 with good taste by knocking out Bsr-d1 and Pi21 genes

稻瘟病是由稻瘟病真菌引起的水稻重要病害,严重影响稻米的产量与品质,培育广谱持久抗性水稻品种是控制稻瘟病最经济有效的措施。利用CRISPR/Cas9技术对江苏主推优良食味粳稻品种南粳9108中2个抗稻瘟病数量性状基因Bsr-d1和Pi21进行敲除研究,获得各基因的纯合敲除系,并通过杂交聚合获得携带双基因敲除系。苗期多菌株接种及叶片划伤接种结果表明,与野生型相比,Bsr-d1和Pi21各单基因敲除系均显著增强稻瘟病抗性;双基因聚合系的抗性水平总体强于各单基因系。RT-qPCR结果显示,稻瘟病菌接种后,多个防御相关基因在敲除系中表达水平显著高于野生型。与野生型相比,Bsr-d1和Pi21编辑系在主要农艺性状、经济和品质相关性状上无显著差异。这一研究创建的抗稻瘟病育种中间材料可进一步用于广谱持久抗稻瘟病优良食味粳稻新品种育种。

Rice blast,caused by Magnaporthe oryzae,is a one of serious diseases in rice,causing huge yield loss and bad quality annually.Breeding rice variety with broad spectrum and durable resistance to blast is the most economical and effective approach to control the disease.In this study,CRISPR/Cas9 technology was used to knock out Bsr-d1 and Pi21 genes,two quantitative resistance genes to blast,in a blast susceptible variety of Nanjing 9108,a well-known japonica rice variety with good taste in Jiangsu province.The homozygous knockout lines for each of the two genes were obtained,and through pyramiding,a double gene knockout line was developed either.Results of spraying inoculation in seedling stage and leaf scratch inoculation with multiple strains showed that,compared with the wild type,knockout lines of either Bsr-d1 or Pi21 all significantly enhanced blast resistance.Resistance levels of double-gene knockout lines were generally stronger than those of the individual gene knockout lines.RT-qPCR results showed that the expression levels of several defense-related genes were significantly higher in knockout lines than those of in wild type.Compared with the wild type,bsr-d1 and pi21 knockout lines showed no significant differences on major agronomic,economic and quality related traits.The new developed lines with enhanced blast resistance are of great value for further breeding new japonica rice varieties towards broad-spectrum and durable resistance to blast and good eating quality.