The Application of CRISPR/Cas9 System in the Preparation ofMutations of Rice Receptor Like Kinase Gene OsBAK1L

Journal Article / 2018

Han, Jiaojiao; Yu, Junping; Song, Ming; Liang, Wanqi

Plant receptor like kinases play an important role in plant growth and the response to external environmental signal stimulation. In order to further understand the function of rice receptor like kinase OsBAK1L, this research used CRISPR/Cas9 technique to edit this gene. OsBAK1L was located in cell membrane and its encoding peotein contained 3 functional domains, including the extracellular LRR domain, the transmembrane domain, and the intracellular kinase domain. In order to further study the function of the different domains of OsBAK1L, two target sequences located in the extracellular LRR domain and intracellular kinase domain respectively were selected as the guide sequences. The synthetic gRNA target sequences were inserted into the entry vector CH, which were then recombined with the expression vector Cas9. The recombinant vectors were transformed into the wild type rice plants 9552 via the Agrobacterium tumefaciens-mediated method. We got 26 and 12 hygromycin resistant transgenic plants for the two constructs respectively. The sequencing analysis for transgenetic plants showed that the introduced construction of CRISPR/Cas9 caused heterozygous sequence change in 3 and 2 T0 plants. Homozygous plants were obtained by the self-crossing of the T0 transgenic line carried 5 bases deletion in the extracellular LRR domain, and the other one carried a base insertion heterozygotes. One in the intracellular kinase domain, both of which caused the gene transcript translation of OsBAK1L to end prematurely. These mutant plants might provide important and stable genetic resource for the further study of the gene function.