作　　者： (刘维）; (刘浩）; (董双玉）; (古丰玮）; (陈志强）; (王加峰）; (王慧）;

机构地区： 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： 《华北农学报》 2017年第4期42-48,共7页

摘　　要： CRISPR/Cas9系统可对植物的内源基因进行有效定点编辑,为作物的遗传育种提供了新的方向。以感病水稻品种丽江为材料,抗病基因OsCOL9为靶基因,探索该系统对水稻内源基因定点编辑效率以及获得有研究意义的突变体。OsCOL9基因CDS序列全长1 266 bp,编码一个422 aa蛋白,分子量大小约为45.6 k Da,蛋白质结构的N端含有B-box结构域,C端含有CCT(CONSTANS、CONSTANS-Like、TOC1)结构域。设计4个长20 nt的guide RNAs(gRNAs)靶点,靶向编辑OsCOL9基因的CDS起始区域以及外显子的末端,分别由U3、U6a、U6b以及U6c启动子驱动,提取T1转基因植株的基因组DNA并对编辑位点附近的DNA片段进行序列分析。结果表明,T1材料中OsCOL9的碱基缺失数最多可达59 bp,突变次数最多可达11次,取得较好的基因编辑效果。同时本试验出现了脱靶效应,因此着重探讨了降低脱靶效应的方法。由于CRISPR/Cas9系统在进行基因编辑时没有外源基因的导入,加上该技术的快捷、简便,对生物技术与传统育种的结合具有重要实践意义。 The CRISPR/Cas9 system can directly to edit endogenous genomic loci in plant,provide a new opportunities for crop breeding. To explore the editing efficiency of the system on rice endogenous gene and obtain meaningful mutants,using the susceptible rice variety Lijiang and the disease resistance gene OsCOL9 as the target gene. The fulllength CDS of OsCOL9 was 1 266 bp long and encoding a 422 aa protein with a molecular weight of about 45. 6 k Da,and the N-terminus contained the B-box domain,and the C-terminus contains the CCT（ CONSTANS,CONSTANS-Like,TOC1） domain. In this study,four 20 nt guide RNAs（ gRNAs） which were targeted to the initiation region CDS and terminal exon of OsCOL9 were designed and transcribed from the U3,U6 a,U6b and U6 c promoters,respectively. The sequences near the editing site were analyzed by extracting genomic DNA from T1 transgenic plants. The number of target base deletions was 59 bp and the number of mutations was up to 11 times. In this study,we get better gene editing results. However,there have been more serious off-target effects in this experiment. Therefore,we focus on off-target effects in the discussion section of this paper. Owing largely to its flexibility,efficiency and no insertion of exogenous gene,it has important practical significance to combine biotechnology and traditional breeding.

关 键 词： 水稻 基因编辑