Mutation Of Wide-compatibility Genes Using TALENs Technology In Rice (Oryza Sativa L.)

Application of rice wide compatibility resources and genes is an effective way of overcoming hybrid sterility and using strong heterosis between indica and japonica of rice (Oryza sativa L.). We conducted a study on the genome edition of rice wide-compatibility gene(S5, S4) and the fragrance gene (OsBADH2) using the Transcription Activator-like Effector Nuclease (TALENs) technology in this paper. A system of TALENs technology, which includes target site design, TALEN vector construction, genetic transformation, differentiation and sequence analysis of target gene, was established in rice. The S4 gene and OsBADH2 gene of Taijing No.9, an elite japonica variety, and the S5 gene of Minghui 86, an elite indica variety, was successfully mutated by the TALENs technology. Mutation rates of the three target genes in the transgenic plants are 84.6%,31.6% and 86.7%, respectively. Using second genetic transformation, we produced some three genes mutant of Taijing No.9, which carried mutations of the S4,OsBADH2 and DST genes (a gene controlling grain number per panicle in rice). Preliminary analysis of phenotype showed that the mutation of OsBADH2 gene could significantly improve the flavor of Taijing No.9.In addition, we found that the exogenous TALEN protein might be mainly expressed in the resistant callus stage, because the subculture of the resistant calli could promote on the continuous multiple edition of the target sequence with exogenous TALEN protein; while the target sequence remain stable in the vegetative growth stage, reproductive development stage and the offspring of the regenerated plants of these resistant calli. Therefore, we established an effective method of producing different mutants of target gene by continuous subculture and differentiation of TALENs transgenic resistant calli. Taken together, the result is not only useful for producing various types of mutants of the target gene, it is but also benefit to promote the application of TALENs technology in rice genetic improvement.