【摘要】 为研究植物激素生长素在模式作物水稻中的功能,我们利用CRISPR-Cas9基因编辑技术,设计水稻生长素合成基因Os YUCCA1的两个靶位点,构建Os YUCCA1基因的敲除载体。通过对Os YUCCA1基因序列分析,将合成的靶点序列插入含hsp Cas9n的载体中,再与p CAMBIA1300重组,构建基因编辑载体,通过农杆菌介导的方法转化水稻品种9522。从78棵转基因苗中鉴定得到针对Os YUCCA1的3种突变类型,包括26棵在外显子第260号碱基处插入碱基A或T两种类型纯合突变和22棵在外显子第447号碱基处插入A、444号碱基C被T替换、445号碱基G被C替换的杂合突变类型。通过分析,发现3种突变株系都存在移码现象而使氨基酸提前终止致使基因突变。本研究成功利用CRISPR-Cas9基因编辑技术敲除了水稻Os YUCCA1基因,为进一步研究Os YUCCA1基因功能提供了理论参考依据。更多还原

【Abstract】 To investigate the role of auxin in model monocot crop rice(Oryza sativa),we constructed two vectors targeting to mutate rice auxin synthetic gene Os YUCCA1 via CRISPR/Cas9 system.The two targeted site sequences of Os YUCCA1 were synthesized and inserted into the vector called hsp Cas9 and then linked to the binary vector of p CAMBIA1300.The resultant plasmids were transferred into rice through Agrobacteriummediated transformation.Three types of mutations were identified from 78 transgenic plants,including 26 homologous plants with A or T insertion respectively at 260 th base on the exon and 22 heterogeneous plants with A insertion at 447 th,C replaced by T at 444 th and G replaced by C at 445 th on the exon.Moreover,the three types of mutations of Os YUCCA1 caused a premature stop and functional loss protein in the homologous mutants.The knockout mutants of Os YUCCA1 created by CRISPR/Cas9 provides the theoretical basis for further study on the role of auxin in rice.更多还原