When cultivated rice seed fall into fields, they may overwinter and spontaneously germinate the next spring. Such germinated plants are termed “volunteer rice.” Volunteer grains originating from feed rice varieties may differ in certain traits, such as quality and taste, as compared with those of rice cultivated for human consumption, which may reduce the overall quality of the final harvested grain. Many rice varieties show resistance to benzobicyclon (BBC), a beta-triketone herbicide (bTH) that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Recently, the rice gene HIS1 (HPPD INHIBITOR SENSITIVE 1) conferring resistance to BBC and other bTHs was identified. In this study, to suppress the occurrence of volunteer rice infestation, we attempted to generate a BBC-sensitive rice strain via the knockout of the HIS1 gene using genome editing techniques. The production of a his1 knockout line was carried out by the start-codon substitution or stop-codon creation using CRISPR-Cas9 cytidine deaminase fusion, which is useful as a novel amino acid sequence is not generated due to the shifting of the reading frame. The mutation frequencies of independent transgenic plants were 3.6, 13.5, 13.8, and 21.2% at four gRNAs for start-codon substitution and three stop-codon creations. The his1 knockout lines were conferred with sensitivity to BBC, re-confirming by genome editing that this is indeed the gene responsible for BBC resistance/sensitivity. The his1 knockout lines also exhibited a sensitive phenotype to other bTHs, including sulcotrione, mesotrione, tembotrione, and tefuryltrione, compared with the wild-type variety ‘Nipponbare.' These results demonstrate the potential of herbicide-sensitive rice produced by genome editing technology as a material to control volunteer feed rice using pre-labeled herbicides for varieties consumed by humans.

当栽培的水稻种子落入田地时，它们可能越冬并在第二年春天自发发芽。这种发芽的植物被称为“志愿者水稻”。与供人类食用的稻米相比，来自饲料稻米品种的志愿者谷物在某些特性（例如品质和口味）上可能有所不同，这可能会降低最终收获谷物的整体质量。许多水稻品种显示出对苯并双环酮（BBC）的抗性，BBC是一种抑制4-羟苯基丙酮酸双加氧酶（HPPD）的β-三酮除草剂（bTH）。最近，水稻基因HIS1 （HPPD抑制剂敏感性1）鉴定出对BBC和其他bTH的抗性。在这项研究中，为抑制自愿性水稻侵染的发生，我们试图产生一种对BBC敏感的水稻菌株通过 淘汰赛 HIS1基因使用基因组编辑技术。生产一个他的1通过使用CRISPR-Cas9胞苷脱氨酶融合的起始密码子替换或终止密码子创建来进行敲除品系，这是有用的，因为由于阅读框的移位而不产生新的氨基酸序列。在用于起始密码子替换和三个终止密码子创建的四个gRNA处，独立转基因植物的突变频率分别为3.6％，13.5％，13.8％和21.2％。的他的1敲除品系被赋予对BBC的敏感性，通过基因组编辑再次确认这确实是导致BBC抗性/敏感性的基因。的他的1与野生型变种“日本晴”相比，基因敲除品系还表现出对其他bTH的敏感表型，包括磺胺三酮，甲基磺草酮，替康三酮和四氢呋喃酮。这些结果证明了通过基因组编辑技术生产的除草剂敏感性水稻的潜力，该材料可以使用预先标记的除草剂控制人为食用的水稻品种的自愿饲料。