摘要
在真核生物中,HORMA结构域具高度保守性,含HORMA结构的蛋白在细胞减数分裂中具有重要作用。LOC_Os01g07170蛋白具有HORMA结构域,系统进化树分析表明该蛋白与拟南芥ASY1相似度较高,且该基因在生殖器官大量表达,推测LOC_Os01g07170基因可能参与细胞的减数分裂。本研究利用CRISPR-Cas9技术对LOC_Os01g07170基因进行敲除,在该基因上设计了两个靶位点,构建LOC_Os01g07170基因敲除载体,采用农杆菌介导的方法转化水稻粳稻品种9522,获得T0代转基因苗。结果显示突变靶点1处共获得7棵转基因苗,鉴定得到3株纯合突变体,突变类型为在第3个外显子的第1 137号碱基缺失1个C碱基;突变靶点2处共获得11株转基因苗,有4株纯合突变体,突变类型为在第4个外显子的第1 501号碱基处插入1个C碱基。分析氨基酸序列,发现这2种突变株系都存在移码突变而使氨基酸翻译提前终止。本研究成功利用CRISPR-Cas9基因编辑技术获得了水稻LOC_Os01g07170基因的两个突变类型的株系,为进一步研究LOC_Os01g07170基因功能提供了遗传材料。
The HORMA domain is highly conservative in eukaryotes. Proteins containing HORMA structure play an important role in cell meiosis. LOC_Os01 g07170 protein contains HORMA domain, and phylogenetic tree analysis indicates that LOC_Os01 g07170 is similar to Arabidopsis ASY1, and LOC_Os01 g07170 is highly expressed in reproductive organs, suggesting that LOC_Os01 g07170 may participate in cell meiosis. In this study,the LOC_Os01 g07170 gene was knocked out by CRISPR-Cas9 technique. Two target loci were designed to construct the knockout vector of LOC_Os01 g07170 gene. Agrobacterium-mediated transformation of rice variety9522 was carried out to obtain transgenic seedlings of T0 generation. The results showed that 7 transgenic seedlings were obtained at the mutation target 1, and 3 homozygous mutants were identified. The mutation type was one C deletion at 1 137 th base on the 3 rd exon. A total of 11 transgenic seedlings were obtained at 2 mutation targets,including 4 homozygous mutants with one C base inserted into the 1 501 th base of the 4 th exon. The amino acid sequence analysis found that there were frameshift mutations in 2 mutant lines, leading to early termination of amino acid translation. In this study, two mutant lines of LOC_Os01 g07170 gene in rice were successfully obtained by CRISPR-Cas9 gene editing technique, which could provide genetic material for further study of LOC_Os01 g-07170 gene function.
引文
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