《植物生理学报》 2017, 53(6): 969-978

利用CRISPR-Cas9系统进行水稻OsMADS15基因的定向编辑

宋凡, 李全林, 鲁丹, 王丽, 袁政^*

上海交通大学生命科学技术学院, 上海200240

通信作者：袁；E-mail: zyuan@sjtu.edu.cn

摘　要：

CRISPR-Cas9系统的发现和应用为快速、定向的植物基因组编辑、突变体创制和开展植物功能基因组研究提供了新的技术工具。在本研究中, 我们利用CRISPR-Cas9系统, 对水稻A类MADS-box转录基因OsMADS15进行定点基因组编辑。结果显示, 该体系在T₀代即获得3个不同的9522^Osmads15双等位突变株系: 株系9522^Osmads15-1等位1在靶点缺失1个碱基A, 等位2在靶点缺失3个碱基ACA; 株系9522^Osmads15-2等位1在靶点缺失5个碱基CAACA, 等位2在靶点缺失3个碱基CAA; 株系9522^Osmads15-3等位1在靶点缺失1个碱基A; 等位2发生了大片段的替换。所有株系等位1的突变均造成了开放阅读框移码, 导致翻译提前终止; 等位2的突变也使氨基酸序列发生不同程度的变化。初步的表型分析显示, 9522^Osmads15突变体小穗不育外稃变长如叶状, 并表现出生殖生长向营养生长的转变; 与已报道的中籼3037^dep突变体表型不同, 除9522^Osmads15-2株系外, 其他2个株系内稃及生殖器官也有向叶片转换的趋势。qRT-PCR分析显示, 3个突变株系的OsMADS15转录本水平显著降低。因此, 本研究对OsMADS15基因第一外显子区域的靶点进行了高效的定点编辑, 获得的9522^Osmads15突变体为进一步开展Os-MADS15调控水稻小穗发育的遗传学和调控网络研究提供了新的遗传材料。

关键词：水稻; CRISPR-Cas9; 基因组定点编辑; 花器官; OsMADS15

收稿：2017-04-10   修定：2017-05-16

资助：国家自然科学基金面上项目(31470397和31671260)和上海市科委基础研究重大重点项目(14JC1403901)。

Genome editing of rice OsMADS15 gene by using CRISPR-Cas9 system

SONG Fan, LI Quan-Lin, LU Dan, WANG Li, YUAN Zheng^*

School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

Corresponding author: YUAN Zheng; E-mail: zyuan@sjtu.edu.cn

Abstract:

The CRISPR-Cas9 system has been developed and applied widely for plant targeted genome editing, mutant creation and functional genomics research. In this work, we applied the CRISPR-Cas9 system to targeted editing of OsMADS15 gene, one of rice A-class MADS-box transcriptional factors, and we have successfully obtained 3 different biallelic lines of 9522^Osmads15 mutants in the T₀ generation. Among them, the 9522^Osmads15-1 line has a single A deletion in biallele-1 and 3 bp deletion in biallele-2; the 9522^Osmads15-2 line has CAACA and CAA deletion in biallele-1 and biallele-2 respectively. Biallele-1 also has a single A deletion and a large fragment replacement was found in the biallele-2 of 9522^Osmads15-3 line. In all of the three obtained T₀ plants, all the biallele-1 had frame-shift mutations in the OsMADS15 open reading frame, resulted in premature translation termination, and all the biallele-2 also had different affects in the translation of OsMADS15. And these mutants grew longer and leafy-like empty glumes, showing transition from reproductive growth to vegetative growth compared to that of wild type. Furthermore, inconsistent to previous phenotypes reported in Zhongxian 3037^dep mutant, all the 9522^Osmads15 mutant lines had longer palea and grew leafy-like reproductive flower organs except in 9522^Osmads15-2. The transcription levels of OsMADS15 were found to be down-regulated obviously in all these mutants by qRT-PCR analysis. Therefore, our research have successfully created 9522^Osmads15 mutants by using CRISPR-Cas9 targeted genome editing system, and provided new materials for further genetic and regulatory network research on OsMADS15 function in rice spikelet development.

Key words: rice; CRISPR-Cas9; targeted genome editing; floral organs; OsMADS15