基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建

摘要

A set oftgw6 (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 ofTGW6were designed and transcribed from the U3, U6a, or U6b promo-ters, 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) byAgro-bacterium-mediated transformation. Sequencing for the genomic DNA ofTGW6locusinT0 rice showed the mutagenesis fre-quency forTGW6was more than 90%, including 51% of homozygous deletion mutations. Further analysis for the T1 mutants showed that almost all the homozygous deletion mutants improved the thousand-grain weight significantly (more than 5%). The successfultgw6 editing not only provided a series oftgw6mutants 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.%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。