Construction of Dwarf1 Mutants in Zhonghua 11 (Oryza sativa L.) by CRISPR/Cas9

Abstract

【Objective】Heterotrimeric G protein α subunit in rice (Oryza sativa L.) plays important biological roles in plant growth and stress response. Null mutants of Gα protein encoding gene DWARF1 (D1) in Zhonghua 11 (japonica cv.) are created, with a view to providing materials for the studies of functional genomics in rice.【Method】sgRNA was designed to target the exon at D1 locus in Zhonghua 11, and CRISPR/Cas9 vector was constructed to transform the embryonic calli of Zhonghua 11. After selection by adding hygromycin, the calli were induced to differentiate and the regenerated plants were identified.【Result】Three mutant lines harbouring different D1 alleles were identified. Real-time quantitative PCR showed a remarkable decrease in D1 mRNA level, suggesting that nonsense-mediated mRNA decay was also triggered. Progenies without hygromycin resistance or vector backbone were further identified, representing transgene-free and stably inherited d1 mutants. According to the observation results of the growth and development of d1 mutants, d1 mutants in Zhonghua 11 showed significantly shorter plant height and smaller panicles. The plant height and panicle length were only 50%-60% compared with those of the wild-type Zhonghua 11. Smaller and rounder seeds were also shown in d1 mutants, meanwhile, the length/width ratio and 1000-grain weight were decreased by about 40%.【Conclusion】Effective frame-shift d1 mutants in Zhonghua11 are obtained, Which creates the genetic materials for functional studies of G protein and other related genes in the future.

Keywords

• gα subunit
• rice
• zhonghua 11
• mutant
• crispr-cas9