利用CRISPR/Cas9定点编辑技术创建水稻长链基激酶突变体oslcbk1和oslcbk2

Construction of Long Chain Base Kinase oslcbk1 and oslcbk2 Mutants in Rice by CRISPR/Cas9 Gene Editing Technique

环境低温严重影响水稻(Oryza sativa L.)的产量和品质。本实验室前期研究发现,与真核细胞膜鞘磷脂代谢相关的长链基激酶LCBKs(Long chain base kinases)和植物的冷适应密切相关。为了进一步解析LCBKs激酶在水稻中的生物学作用,本研究利用基因组定点编辑技术CRISPR/Cas9,构建了水稻OsLCBK1和OsLCBK2的单基因和双基因编辑载体。通过农杆菌EHA105介导的愈伤转化法侵染粳稻‘中花11’,抗性筛选获得阳性植株。结合测序分析发现,T0代的转基因植株靶标位点发生了多种类型的突变,包括纯合、杂合和双等位等。本研究成功实现了OsLCBK1和OsLCBK2的单基因及双基因定点编辑,获得了多种突变类型的水稻长链基激酶单突变体oslcbk1、oslcbk2以及双突变体oslcbk1&oslcbk2。这些突变体为揭示水稻Os LCBKs响应低温应答以及参与其他生物学过程提供了研究材料。

Rice(Oryza sativa L.)is an important commercial and warm-season crop.Low temperature can reduce quality and production of rice plants during any developmental stage.Previous studies have found that the Ara-%bidopsis LCB kinases(LCBKs)play a prominent role in plant chilling and freezing tolerance.To further explore the biological function of OsLCBK1 and its homology gene OsLCBK2 in rice,the technology CRISPR/Cas9 was used to edit their genome.The recombinant plasmids were transferred to a rice cultivar’Zhonghua11’by Agrobac-%terium-mediated transformation method.Hygromycin-resistance screening and DNA sequencing analysis revealed that many types of mutations occurred in the target sites of T0 transgenic plants,including homozygous mutation,biallelic mutation and heterozygous mutation.Our results indicated that the s LCBK1 and OsLCBK2 gene was edited successfully by CR ISPR/Cas9 technique.A variety of LCBK single mutant oslcbk1,oslcbk2 and double mutant oslcbk1&oslcbk2 were obtained.These genetic materials were provided to further explore the biological functions and molecular mechanisms of OsLCBKs in the process of rice chilling tolerance.