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Maste

CRISPR/Cas9 매개 돌연변이 유발을 통한 자스모네이트-ZIM-도메인 단백질 9의 특성 규명

학위논문 (석사)-- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2018. 8. 최양도.CRISPR/Cas9 system is a powerful technique for genome editing. It is more effective and convenient than any other genome editing methods. In this study, I generate JAZ9 knock-out mutant rice by using CRISPR/Cas9 system. The guide RNA was recombined with the rice U3 promoter in the CRISPR/Cas9 binary vector pRGEB31 carrying the gRNA scaffold III. The guide RNA sequence was designed from the CRISPR-PLANT website (http://www.genome.arizona.edu/crispr) to avoid off-target effects. Total 26 individual T0 transgenic rice were produced through Agrobacterium-mediated callus transformation. Cas9-mediated mutation was occurred upstream of 3-nucleotide position from protospacer adjacent motif site in the target region. Nucleotide sequence analysis showed that most T0 transgenic plants were genetically chimeric mutants. Just two individual mutants (approximately 8%) were homozygotic bialle chimera at the target site, and 16 individual mutants (approximately 62%) were heterozygotic chimeras. I classified these 26 individual lines into 4-groups according to the mutation pattern type. First, if no mutation occurs, 'only wild-type sequence'. Second, the cases of biallelic homozygotic chimeric mutations are grouped 'only mutated sequences'. Third, when a wild-type sequence and a mutated sequence coexist (codominant), the 'WT and mutated sequence together'. Finally, if the wild-type sequence and the mutated sequence coexist, but the ratio of the mutated sequence is much lower, the 'WT and the weak mutated sequence together'. Some chimeric mutant did not remain in the same group and mutations were observed or disappeared with the developmental stage. The mutation rate tended to increase with higher Cas9 expression level. However, the copy number of Cas9 transgene did not determine the level of Cas9. To obtain additional homozygotic mutant plants, I carried out further analysis with the T1 and T2 siblings. All T1 siblings from T0 mutant plants with homozygotic mutation still maintained homozygotic mutation. I also found that 60% of heterozygotic mutant plants in T0 produce the homozygotic mutant in T1. These suggest that homozygotic mutant can be obtained in T1 generation from T0 mutant of heterozygotic chimeras. The number of individual homozygotic mutant plants, therefore, can be increased through genetic segregation of T0 heterozygotic mutants.Introduction 1 Materials and Methods 3 1. Plant material, transformation and growth. 3 2. Construction of DNA plasmid for Cas-mediated mutagenesis. 3 3. Extraction of rice genomic DNA, PCR and nucleotide sequencing 3 4. Quantitative RT-PCR and TaqMan PCR analysis 4 5. Yeast Two-Hybrid assay 4 6. Site-directed plasmid mutagenesis 5 Results 6 1. Generation of osjaz9 mutant by CRISPR/Cas9 system. 6 2. Genetic chimerism in T0 generation 6 3. Correlation between edited nucleotide and mutation pattern 8 4. The genome editing pattern can vary depending on the developmental stage. 9 5. Further analysis of genome editing pattern shows complexity of groups. 10 6. Cas9 expression level is correlated with the genome editing frequency 11 7. Homozygotic mutant can be obtained in T0 generation. 11 8. Homozygotic mutants were obtained even from T0 heterozygotic mutant. 12 9. Mutated OsJAZ9 protein did not interact with JA receptors. 13 10. OsJAZ9 negatively regulate JA response in rice. 14 Discussion 15 References 18 국문초록 46Maste