基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建  

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

作　　者：王加峰[1] 郑才敏 刘维[1] 罗文龙[1] 王慧[1] 陈志强[1] 郭涛[1] 

机构地区：[1]华南农业大学国家植物航天育种工程技术研究中心,广东广州510642

出　　处：《作物学报》2016年第8期1160-1167,共8页Acta Agronomica Sinica

基　　金：广东省公益研究与能力建设转型项目(20150209);国家高技术研究发展计划(863计划)项目(2011AA10A101);国家现代农业产业技术体系建设专项(CARS-01-12)资助~~

摘　　要：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(g RNA)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p YLCRISPR/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。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（g RNA） targeted to the exon of TGW6 were designed and transcribed from the U3, U6 a, or U6 b promoters, respectively. The three target sites of g RNA were then ligated to the vector p YLCRISPR/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 locus in T0 rice showed the mutagenesis frequency for TGW6 was 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 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.

关 键 词：水稻 基因编辑 CRISPR/Cas9 TGW6 千粒重 

分 类 号：S511[农业科学—作物学]