利用CRISPR/Cas9技术编辑OsOFP30基因创制水稻粒型突变体

Creation of Rice Grain Size Mutants by Editing OsOFP30 via CRISPR/Cas9 System

【目的】研究水稻OFP家族转录因子OsOFP30对粒型的影响,创制新的粒型突变材料,为水稻粒型改良提供参考。【方法】以粳稻品种中花11为受体材料,利用CRISPR/Cas9技术对OsOFP30基因进行定向编辑;通过T_(0)、T_(1)和T_(2)代连续筛选,获得3类无外源片段插入的纯合突变体(长片段删除突变OsOFP30^(-89)、单碱基插入突变OsOFP30^(+1G)和OsOFP30^(+1A));分析粒型变异,进行生物信息学和测序分析,初步确定粒型变异原因。【结果】与野生型相比,3类突变体的粒宽和千粒重都显著降低,OsOFP30^(-89)和OsOFP30^(+1G)仅粒长和粒厚显著降低,穗型指标与野生型无明显差异,OsOFP30^(+1A)的粒长和粒厚与野生型无明显差异,仅一次枝梗数显著低于野生型;生物信息学分析表明,这3类突变体均由移码突变导致的翻译提前终止所产生,OsOFP30^(-89)突变蛋白长度为252个氨基酸,OsOFP30^(+1G)和OsOFP30^(+1A)突变蛋白长度均为282个氨基酸,两者仅第323位的核酸序列存在G和A的单碱基差异,导致突变蛋白第108位产生丝氨酸和天冬酰胺的差别。【结论】初步判断OsOFP30基因可调控水稻粒型变异。本研究创制了新的粒型突变材料,可为水稻粒型改良育种提供参考。

【Objective】To study the role of OsOFP30,a transcription factor of the rice OFP family,in regulating grain size and to create new mutant materials of grain size,thereby providing a new reference for the improvement of rice grain size.【Method】The japonica rice line Zhonghua 11 was utilized as a transgenic recipient to generate OsOFP30 mutants using the CRISPR/Cas9 system.Through continuous screening of the T_(0),T_(1),and T_(2) generations,three types of homozygous mutants without foreign fragment insertions were obtained:the long segment deletion mutation OsOFP30^(-89),the single base insertion mutation OsOFP30^(+1G),and OsOFP30^(+1A).Grain size was measured at maturity stages,and the potential reasons for the different grain sizes were analyzed through bioinformatics and sequencing.【Results】Compared with the wild-type,the grain width and 1000-grain weight of all three types of mutants were significantly reduced.For OsOFP30^(-89) and OsOFP30^(+1G),only the grain length and grain thickness are significantly reduced,with no obvious differences in panicle shape indicators from the wild type.The grain length and grain thickness of OsOFP30^(+1A) show no obvious differences from the wild type,and only the number of primary branches is significantly lower than that of the wild type.Bioinformatics analysis revealed that the three types of mutants introduced premature translation termination due to a frameshift mutation.The OsOFP30^(-89) mutant protein consists of 252 amino acids,while the OsOFP30^(+1G) and OsOFP30^(+1A) mutant proteins consist of 282 amino acids.Sequence analyses showed that OsOFP30^(+1G) and OsOFP30^(+1A) differ by a single base,with G and A at the 323rd position,respectively.This variation resulted in a change between serine and asparagine at the 108th position of the mutant protein.【Conclusion】The OsOFP30 gene plays a crucial role in regulating rice grain size.This study successfully created new mutant materials affecting grain size,which will provide valuable references for the improvement of rice grain size.