Targeted Editing of OsIDQ1 Gene in Rice Based on CRISPR /Cas9 Technology

Objective IQD gene family proteins are plant specific calmodulin-binding target proteins, which play an important role in plant defense and development regulation. The interaction between Pik-H4 and OsIQD1 was found in the yeast double hybrid screening experiment in laboratory. In order to reveal the specific biological functions of OsIQD1 in rice blast resistance, OsIQD1 gene was edited by using CRISPR/Cas9 editing technique.

Method OsIQD1 was obtained by sequence alignment. Two 20 bp editing targets were designed in exon 1 and exon 5 of OsIQD1 (DUF4005 domain), and the nucleotide fragments of the two targets were cloned into pRGEB32 vector to obtain pRGEB32-OsIQD1 -gRNA vector. The rice Pik-H4 NIL callus was transformed by agrobacterium-mediated method. Transgenic positive plants were obtained by regeneration culture and hygromycin resistance screening. PCR and sequencing were performed on the target region sequences of transgenic plants of T₀ generation to analyze the mutation types of osiqd1.

Result The transgenic regenerated plants were identified by PCR with hygromycin resistance gene HPT Ⅱ, and 30 positive lines were obtained. Sequencing analysis of the sequences near the target site of T₀ generation plants showed that OsIQD1 gene was successfully edited, and the editing efficiency of the two sites was 21.67% and 26.67%, respectively. There was one homozygous mutant line in target 2 region.

Conclusion The study results provide genetic material for further studies on the mechanism of osiqd1 mutation involved in calcium ion/calmodulin signaling pathway, and OsIQD1 is preliminarily presumed to be involved in the basic immune response of plants.