Construction of Rice Mutants of the Polyamine Oxidase Gene OsPAO4 Based on CRISPR/Cas9 Editing

Abstract

【Objective】Rice blast is an important limiting factor in rice production. Exploring and utilizing resistant genes is an important guarantee for achieving high and stable yield of rice. Previous studies have shown that rice blast resistance-related microRNA osa-miR-21 may regulate rice blast resistance by targeting the polyamine oxidase gene OsPAO4. However, the function of Polyamine Oxidases (PAOs) in rice disease resistance has not been reported. In order to further explore the possible biological function of OsPAO4 gene in rice disease resistance, the CRISPR/Cas9 editing technology was used for site-specific editing of ospao4 gene mutants and the edited offspring were also analyzed.【Method】A 20-bp editing target was designed in the second exon of OsPAO4, and the nucleotide fragments of the target was cloned into pRGEB32 vector to obtain OsPAO4 editing vector. Then, the rice Pik-H4 NIL callus was transformed by Agrobacterium-mediated method. Positive transgenic plants were obtained by regeneration culture and hygromycin detection, PCR and sequence analysis were performed on the DNA sequences near the target site in the T0 generation plants.【Result】The results show that the OsPAO4 gene was successfully edited and finally 25 transgenic-positive plants were obtained, and various mutation types were produced in the T0 generation, including 3 homozygous mutants, 18 heterozygous mutants and 4 nonedited plants. In addition, the glume color of the heterozygous mutation ospao4-8 of the T0 generation changed from purple to dark gray, and the activity of catalase (CAT) in the mutants was increased. It was predicted that OsPAO4 might participate in the basis immunization process.【Conclusion】A variety of ospao4 mutants were created in this study, and it was preliminarily determined that OsPAO4 might participate in the regulation of rice immune process, which laid an important foundation for further revealing the specific biological function and molecular regulation mechanism of OsPAO4 gene.

Keywords

• rice
• ospao4
• crispr/cas9
• gene editing
• polyamine oxidase