摘要： 为研究水稻生长素响应因子OsARF12在水稻生长发育中的作用，通过CRISPR/Cas9基因编辑技术，设计OsARF12 2个不同外显子突变靶点，化学合成包括突变靶点的特异性序列，并与中间载体sgRNA连接进而与Cas9终载体重组获得OsARF12 2个不同突变靶点的表达载体。选取测序成功的单一克隆，利用农杆菌介导法导入粳稻品种日本晴，获得转基因植株，并结合测序结果分析突变单株的突变类型，T₁突变体KO-ARF12-1有3种纯合突变、2种双等位突变，共5种突变类型；KO-ARF12-2有6种纯合突变、4种双等位突变和1种杂合突变，共11种突变类型。挑选突变类型为缺失1 nt、缺失4 nt、缺失7 nt和缺失2 nt插入1 nt、缺失5 nt的株系作为研究对象，荧光定量PCR鉴定结果表明，OsARF12在突变体中的表达量较野生型极显著下降（P<0.01）。在正常大田生长条件下，2种突变体不同突变类型株系与野生型相比，株高显著降低，降幅为13.15%~18.39%；第1，2茎节间长度无显著差异，第3，4茎节间长度显著降低，降幅为21.91%，23.46%。利用CRISPR/Cas9创制的OsARF12突变体，对水稻节间延伸及株高具有一定调节作用，对于完善水稻生长素响应因子的分子调控机制具有重要意义。

关键词: 水稻, OsARF12, CRISPR/Cas9, 基因编辑, 株高

Abstract: In order to study the role of rice auxin response factor OsARF12 in the growth and development of rice, two different exon mutation targets of OsARF12 were designed by CRISPR/Cas9 gene editing technology, respectively. The specific sequences including mutation targets were synthesized and linked with intermediate vector sgRNA, and then recombined with the final vector Cas9 to obtain two independent expression vectors with different mutation targets. The single clone which was sequenced successfully was introduced into japonica rice variety Nipponbare by Agrobacterium tumefaciens mediated method to obtain transgenic plants, and the mutation types of mutant plants were analyzed by sequencing results. There were three homozygous mutations and two biallelic mutations in T₁ mutant KO-ARF12-1, with a total of five mutation types; There were 6 homozygous mutations, 4 biallelic mutations and 1 heterozygous mutation in KO-ARF12-2. The mutant lines with deletion of 1 nt, deletion of 4 nt, deletion of 7 nt and deletion of 2 nt, insertion of 1 nt, deletion of 5 nt were selected as the research objects. The results of fluorescence quantitative PCR showed that the expression of OsARF12 in the mutants was significantly lower than that in the wild type. Compared with the wild type, the plant height of the two mutants with different mutation types decreased significantly, with a decrease range of 13.15%-18.39%; There was no significant difference in the length of the first and second internodes, but the length of the third and fourth internodes decreased significantly by 21.91% and 23.46%. The OsARF 12 mutant created by CRISPR/Cas9 can regulate the internode extension and plant height, which is of great significance to improve the molecular regulation mechanism of auxin response factor in rice.

Key words: Rice, OsARF12, CRISPR/Cas9, Gene editing, Plant height

中图分类号: 

• S511.03

• Q78