Abstract：Abstract Low temperature is one of the major environmental factors that influence rice (Oryza sativa) growth, development and production. Rice seedlings are particularly sensitive to chilling in early spring. Therefore, improvement of chilling tolerance in rice may significantly increase rice quality and production. To identify genes participated in the regulation of plant chilling tolerance, the cold-resistant gene library was constructed through suppression subtractive hybridization (SSH) in a previous study. From the SSH library, a transcription factor TIFY1b (LOC_Os03g52450) with an conserved TIF[F/Y]XG domain, which might involve in the rice cold resistance, was isolated. To explore the function of TIFY1b and its homology gene TIFY1a (LOC_Os03g47970), the technology of “clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)” was used to edit their genome. According to the characteristics of CRISPR/Cas9 editing system and the conservation of target genes (TIFY1a and TIFY1b), 2 sites of 20 nt guide RNA (gRNA) targeted to the exon of TIFY1a were designed and named A1 and A2; 2 sites of 20 nt gRNA targeted to the exon of TIFY1b were designed and named B1 and B2. A1, A2, B1 and B2 were all ligated and constructed individually to the CRISPR/Cas9 vector with single target site to create tify1a or tify1b single mutant; A1 and B1 were together ligated and constructed to another CRISPR/Cas9 vector which harbors 2 target sites to create tify1a & tify1b double mutant. Then, these recombinant plasmids were all transferred to a rice cultivar Nipponbare by Agrobacterium-mediated transformation method. T0 transgenic mutants were obtained and confirmed by hygromycin-resistance screening. Further sequencing for the genomic DNA of TIFY1a or TIFY1b gene locus in T0 transgenic lines showed 60% mutagenesis frequency in target A1 site, 87.5% mutagenesis frequency in target A2 site, 35% mutagenesis frequency in target B1 site, 75% mutagenesis frequency in target B2 site, and 62.5% mutant ratio of lines containing both target A1 and target B1. These results suggested that CRISPR/Cas9 systems can effectively induce site-specific mutations in T0 rice plants. Moreover, the results found various targets of mutant types presented in the T0 transgenic mutants, such as deletion of bases, insertion of bases, insertion behinds deletion of bases, and long fragment deletion of genomic DNA. The major mutation type in this study was 1 bp insertion and 1 bp deletion mutation. In addition, The results found that the positions of the mutation could occur in the upstream of the proto spacer adjacent motif (PAM) region ranging 4~7 bp, and the mutation occurred in the 4 bp upstream of the PAM region was more often. Consequently, 3 different mutation types mediated by the CRISPR/Cas9 system were formed in T0 transgenic lines, including homozygous mutation, biallelic mutation and heterozygous mutation. All these mutation types could descend stably into the next generation. Furthermore, protein analysis indicated that frameshift or premature of proteins which caused function loss of TIFY1a or TIFY1b gene happened in T0 transgenic mutants. Collectively, a series of different types of tify1 mutant lines were successfully obtained by using CRISPR/Cas9 technology in this study and could be used to investigate the role of TIFY1 genes in rice adaptation to chilling temperature. Our studies might reveal a novel pathway that controls cold adaptation in rice and will help to broaden the possibilities for genetically engineering cold-tolerant rice cultivars.
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