CRISPR-Cas9多靶点载体构建及高效定向编辑水稻苯达松抗性基因CYP81A

A CRISPR-Cas9 Vector System for Multiplex Targeting and Its Application in Editing Bentazon Resistance Gene CYP81A6 in Rice(Oryza sativa)

水稻CYP81A6基因(Bel)编码的细胞色素P450单加氧酶对苯达松和磺酰脲类除草剂具有抗性,该基因的隐性纯合突变体(bel)表现为苯达松敏感致死性,可作为植物化学致死标记,在水稻杂交制种中具有重要的应用价值。本研究在CYP81A6基因的首个外显子中设计两个靶位点,构建了含多靶点的CRISPR-Cas9载体,通过根癌农杆菌介导法转化粳稻中花11,获得了56株转基因T0代阳性植株,提取其中9株基因组DNA进行靶位点测序检测,发现基因敲除突变率为77.8%,其中纯合突变率高达71.4%,对纯合突变体水稻喷施苯达松,均表现出对苯达松敏感致死。为应用CRISPR-Cas9技术培育苯达松敏感致死水稻不育系和对其他水稻基因进行编辑提供理论依据。

The rice cytochrome P450 monooxgenase, which is encoded by CYP81A6 （Bel） gene, confers the resistance to bentazon and sulfonylurea herbicides. The recessive homozygous mutant （bel） of Bel shows bentazon sensitive lethality, which can be used as chemical lethal marker and will have important application values in hybrid rice seed production. In this study, two single guide RNAs were designed based on the first exon within the coding sequence of CYP81A 6 gene. A total of 56 positive transgenic TO plants were obtained, and the genomic DNA of 9 plants were extracted for testing the gene mutation by sequencing. The total gene mutation efficiency was 77.8%, and the homozygous mutation efficiency was up to 71.4%. After spraying bentazon, the homozygous mutants showed bentazon sensitive lethality. These results provided a theoretical basis to use CRISPR-Cas9 system for breeding bentazon-lethal male sterile lines and editing other function genes in rice.