基于CRISPR/Cas9技术的水稻反光敏不育基因csa突变体的获得

Acquisition of Mutants of the Reverse Photoperiod-sensitive Genic Male Sterility Gene csa in Rice Based on CRISPR/Cas9 Technology

利用CRISPR/Cas9技术,以反光敏育性基因CSA为编辑对象,构建了敲除载体pCAMBIA1301-Cas9-CSA-gRNA,用农杆菌介导法转化粳稻WG07,并分析CSA基因的突变类型。T0代转基因苗中有56株CSA突变单株,其中7株为纯合突变,纯合突变类型包括单碱基A、T和G的插入以及1个和3个碱基的缺失。长沙大田种植表明,短日照条件下,T1代无选择标记基因的csa突变体的花粉为完全不育;csa突变体的株高比野生型明显降低,分蘖数与野生型相比明显增加,而穗长与野生型相比无显著差异。粳稻csa突变体的获得为培育粳稻反光敏不育新种质奠定了材料基础。

CSA,a gene related to the reverse photoperiod-sensitive genic male sterility(rPGMS),was selected as the target for genome editing by way of the CRISPR/Cas9 technology.The pCAMBIA1301-Cas9-CSA-gRNA vector was constructed for knocking out CSA,and the vector was transformed into japonica rice variety WG07 through the Agrobacterium-mediated method.The types of csa mutations were analyzed in the transgenic lines.There were 56 mutants in T0 transgenic plants,of which six plants were the homozygous mutants.The homozygous mutations included the single-base insertion of A,T or G,and the deletion of one or three bases.In T1 generation,the pollen fertility of marker-free homozygous csa mutants with single-base insertion showed complete male sterility in the field at Changsha under short daylength conditions.Compared with the wild type,the plant height of the csa mutant was significantly lower and the tiller number per plant increased significantly,but the panicle length had no significant difference.The acquisition of japonica csa mutants lays a material foundation for breeding the rPGMS japonica rice.