基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建

利用CRISPR/Cas9技术对调控水稻产量千粒重基因TGW6定点编辑，获得了一套有重要育种价值的tgw6突变体。设计了分别由U3、U6a和U6b启动子驱动、长20 bp的guide RNA (gRNA)靶点以靶向编辑TGW6基因的外显子，首先将这3个靶点一起组装到pYLCRISPR/Cas9-MT(I)载体上，然后利用农杆菌介导侵染水稻材料H447 (R819/玉针香//R819的BC₃F₆)；提取T₀代转基因植株的基因组DNA并对编辑位点附近的DNA片段进行PCR检测及测序分析。结果表明，T₀代材料中tgw6的突变频率高达90%，其中纯合缺失突变率约占51%。对T₁代纯合缺失突变体的千粒重性状的调查分析结果表明，部分tgw6的缺失突变能显著提高千粒重(大于5%)。不同类型tgw6突变体的成功创建不仅丰富了tgw6的变异类型，为水稻的高产稳产奠定了重要的材料基础，还证实了CRISPR/Cas9技术在水稻基因工程育种中高效、易操作的特点，具有重要的理论与实践意义。

Construction of tgw6 Mutants in Rice Based on CRISPR/Cas9 Technology

A set of tgw6 (Thousand grain weight 6) mutants were constructed using CRISPR/Cas9 technology in this study. Three sites of 20 nt guide RNA (gRNA) targeted to the exon of TGW6 were designed and transcribed from the U3, U6a and U6b promoters, respectively. The three target sites of gRNA were then ligated to the vector pYLCRISPR/Cas9-MT(I) based on golden gate cloning strategy. The recombinant plasmid was transferred to a rice cultivar, H447 (R819/Yuzhenxiang//R819 BC₃F₆) by Agrobacterium-mediated transformation. Sequencing for the genomic DNA of TGW6 locusinT₀ rice showed the mutagenesis frequency for TGW6 was more than 90%, including 51% of the homozygous deletion mutations. Further analysis for the T₁ mutants showed almost all the homozygous deletion mutants improved the thousand grain weight significantly (more than 5%). The successful tgw6 editing not only provided a series of tgw6 mutants for high and stable yield of rice but also proved that CRISPR/Cas9 is a facile and powerful means of rice genetic engineering for scientific and agricultural applications, which has important theoretical and practical significance for rice breeding.