利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状

摘要

[Objective]Gene orientation editing has become an important way for molecular breeding. We evaluated the improvement effects on the target traits following the construction of GS3 and Gn1a loss-of-function mutants so as to lay a solid foundation for high-yielding rice breeding. [[Method]GS3 and Gn1a were selected as targets for gene editing, which control the grain size and grain number in rice, respectively. The Pc1300-2×35S:: Cas9-Ggs3-gGn1a expression vector was constructed for knocking out both GS3 and Gn1a by using CRISPR/Cas9 system, and transformed into four good quality rice varieties by the Agrobacterium-mediated method. And the properties of mutations and the target traits were analyzed in the transgenic lines.[Result]The sequencing results showed the GS3 and Gn1a in four rice varieties were successfully edited. In T0 generation, we obtained mutants with frame shift mutations in GS3 and Gn1a in four rice varieties. In T1 generation, the marker-free plants were screened for analyzing the agronomic traits in four genetic backgrounds. For the agronomic characters, the gs3 and gs3gn1a mutants had the longer grain length and the increased 1000-grain weight compared to the wild type, and the gs3gn1a mutants had more grains per panicle compared to the gs3 mutants.[Conclusion]CRISPR/Cas9-mediated gene editing can improve rice target traits, which has great potential in orientation improvement of rice varieties.%[目的]基因组定点编辑技术已成为分子育种的重要手段.本研究拟对GS3和Gn1a功能缺失突变对目标性状的改良效应进行分析,以期为培育高产水稻提供理论基础.[方法]利用CRISPR/Cas9系统,以控制粒型基因GS3和控制每穗粒数基因Gn1a为编辑对象,构建了共敲除载体pC1300-2×35S::Cas9-gGS3-gGn1a,用农杆菌介导法转化4个优质水稻品种,分析了基因突变的特征和相应农艺性状.[结果]构建的敲除载体成功地实现了对GS3和Gn1a基因的定点编辑.在4个转化受体的T0代均分别获得了gs3和gs3gn1a的移码突变体.对T1代中无选择标记突变体的农艺性状分析表明,突变体gs3和gs3gn1a与野生型相比粒长变长,千粒重增加;突变体gs3gn1a与突变体gs3相比,每穗粒数显著增加.[结论]利用CRISPR/Cas9系统进行水稻基因编辑可以快速改良品种的目标性状,在水稻品种的定向改良方面具有巨大的潜力.