利用CRISPR/Cas9技术定向编辑水稻过氧化氢酶基因OsCAT

CRISPR/Cas9-mediated Editing of the Rice Catalase Gene OsCAT2

过氧化氢酶(catalase,CAT)作为重要的抗氧化酶,可催化细胞内过氧化氢的分解,为了研究水稻过氧化氢酶基因OsCAT2的功能,首先根据在线软件http://crispr.mit.edu/搜寻向导sg RNA序列,选择OsCAT2基因第三外显子上的"GTGGAGAACCGAACAATAACTGG"作为目标靶序列,然后构建了OsCAT2的CRISPR/Cas9载体BGK03-OsCAT2,将目的载体转化水稻粳稻9522后共获得了7个独立的转基因株系,其中包括在PAM附近发生编辑的独立转基因株系2个,编辑方式主要表现为缺失和错配。与野生型相比,转基因植株较矮、分蘖数增加、叶片呈明显的病叶状、成熟时期育性明显降低,这为深入研究水稻OsCAT2基因的作用机制提供了帮助。

As an important antioxidant enzyme, catalase （CAT） can catalyze the decomposition of hydrogen peroxide in cells. In order to investigate the function office catalase gene OsCAT2, we first searched the suitable guide sgRNA sequence using the online software http：//crispr.mit.edu/, and ＂GTGGAGAACCGAACAATAACTGG＂ sequence of the third exon of OsCAT2 gene was selected as the target sequence. Then, the CRISPR/Cas9 vector BGKO3-OsCAT2 of OsCAT2 gene was constructed. Finally, the target vector was transformed into rice japonica 9522 and 7 independent transgenic plants were obtained, including 2 independent transgenie lines edited near PAM, and the editing methods were mainly missing and mismatched. Compared with the wild type, the transgenic plants were shorter, the number of tillers increased, the leaves showed obvious leaf shape, and the fertility of the mature period decreased significantly, which helped to further study the mechanism of the action of rice OsCAT2 gene,