利用CRISPR-Cas9系统进行水稻OsMADS15基因的定向编辑

Genome editing of rice Os MADS15 gene by using CRISPR-Cas9 system

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Os MADS15进行定点基因组编辑。结果显示,该体系在T_0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。q RT-PCR分析显示,3个突变株系的Os MADS15转录本水平显著降低。因此,本研究对Os MADS15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OsMADS15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。

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 Os MADS15 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^Osmads15-1line 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 Os MADS15 open reading frame, resulted in premature translation termination, and all the biallele-2 also had different affects in the translation of Os MADS15. 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 depmutant, 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 Os MADS15 were found to be down-regulated obviously in all these mutants by q RT-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 Os MADS15 function in rice spikelet development.