Rice mutant establishment of the mutated phosphate transporter OsPht gene by the CRISPR/Cas9 gene editing system for evaluating nutrient translocation

ObjectivesThe CRISPR/Cas9 system, a generated adaptive immune system in bacteria and archaea bacteria by defending against the invasion from exogenous DNA fragments, is widely used nowadays as a gene editing tool in many animal and plant gene modification. Phosphorus is an essential nutrient element for plant growth, its absorption is mainly fulfilled through the transmembrane phosphate transporters in plants. In this study, the CRISPR/Cas9 gene editing technology was applied to modify rice phosphate transporter protein OsPht gene sharing the highest homology with the phosphate transporter protein McPht gene in Mesembryanthemum crystallinum, which would provide a reliable material for scientifically elucidating the function of McPht transporter protein.

MethodsThe rice phosphate transporter protein OsPht sharing high homology with McPht protein was edited by CRISPR/Cas9 gene editing system. Briefly, based on the sgRNA mediation, the fragments generated by PCR using a pair of specific primers were inserted to the expression vector pCXUN with U3 promoter, and the resulting transformants were transformed into Agrobacterium EHA105 to infect the rice immature embryos of rice Oryza sativa L. japonica. cv. Kitaake by Agrobacterium transformation method. The T0 generating plant seedlings with anti-antibiotic were transplanted to the soils in field, and the T1 generation seeds were obtained and used for pot culture experiments, and the genomic DNA of rice seedling leaf was extracted for PCR detection, and all the PCR products were purified and sequenced, and the edited sites in the target gene fragments were determined according to the sequence changes, and the physiological measurements of mutant lines were performed.

Results1) The mutants with the bases deletion or substitution in the OsPht gene were divided into two categories, which were represented by separately deleting 6 bases and 8 bases in the target editing fragment with 20 bases. 2) The mutants missing 6 bases accounted for 28.6% of the total mutant lines, and the mutants missing 8 bases accounted for 42.9% of the total mutant lines, and the mutants with mutation of GT bases accounted for 28.6% of the total mutant lines. 3) The plant height, fresh weight and root activity of mutant rice were lower than those of the wild type. The root length, shoot area, surface area and lateral root number were higher than those of the wild type. The contents of chlorophyll and soluble sugar of Cas-2 were higher than those of the wild type, and demonstrated remarkable differences, but the contents of chlorophyll and soluble sugar of Cas-7 were lower than those of the wild type, and no significant difference was observed.

ConclusionsThe CRISPR/Cas9 gene editing system has successfully edited the rice homologous OsPht of the McPht, and these mutants with bases deletion or substitution in the OsPht gene not only establish a reliable materials basis for scientifically elucidating the function of the target gene, but also simultaneously provide valuable material supports, which the phosphate transporter McPht gene is introduced into the mutants with bases deletion or substitution in the OsPht gene, for systematically verifying physiological and biochemical functions of exogenous homologous from other plants. This study shows that the CRISPR/Cas9-mediated gene editing system is a scientific and effective pathway for the functional evaluation of nutrient transporter genes in plant.