표제지
ABSTRACT
목차
1. 서론 10
2. 재료 및 방법 19
2.1. 식물 재료 19
2.2. 총 안토시아닌 추출 및 함량 측정 19
2.3. 총 RNA 추출 및 cDNA 합성 20
2.4. Reverse Transcription-Quantitative PCR (RT-qPCR) 분석 21
2.5. Genomic DNA 추출 23
2.6. 유전자 클로닝 및 염기서열 분석 24
2.7. 계통수 분석 24
2.8. Yeast Two-Hybrid Assay 26
2.9. 식물체 내의 유전자 임시발현 검정 26
2.10. 애기장대 원형질체 분리 및 형질전환 29
2.11. 프로모터 활성 검정 30
2.12. Single Guide RNA (sgRNA) 합성 및 유전자 교정용 운반체 제작 32
2.13. Agrobacterium 매개 배추 형질전환 및 조직배양 32
3. 결과 및 고찰 34
3.1. 초록배추와 빨강배추의 안토시아닌 함량 측정 및 BrMYBL2.1의 발현 분석 34
3.2. BrPAP1, BrTT8과 BrMYBL2.1의 유전자 염기서열 및 억제 도메인 분석 40
3.3. 초록배추와 빨강배추 유래의 BrMYBL2.1과 BrTT8의 상호작용 확인 49
3.4. 담배 잎 임시발현을 통한 BrMYBL2.1의 기능 분석 52
3.5. BrMYBL2.1, BrPAP1 및 BrTT8에 의한 BrCHS와 BrDFR의 프로모터 활성 57
3.6. BrMYBL2.1의 유전자 교정을 통한 안토시아닌 증진 배추 육성 59
4. 결론 65
참고문헌 67
국문요약 73
Table 1. Lists of primers were used in this study. 22
Table 2. Primers for gene cloning. 25
Table 3. Primers for Y2H construct. 27
Table 4. Primers for trasnsient assay constructs. 28
Table 5. Primers for promoter activation assay. 31
Table 6. Media components for Chinese cabbage transformation. 62
Table 7. Analysis of In-Del frequency at BrMYBL2.1 transgenic Chinese cabbage. 64
Figure 1. Anthocyanin biosynthesis pathway in plants. ANS, anthocyanidin synthase; AT, acyltransferase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase;... 12
Figure 2. Putative regulation model for anthocyanin biosynthesis. (A) Activation of anthocyanin biosynthetic genes by the active MBW complex. (B) Inhibition of anthocyanin biosynthetic genes by... 13
Figure 3. Conservative characteristics of MYB proteins that positively or negatively regulate anthocyanin biosynthesis. Motifs are labeled with the following abbreviations: EAR, EAR-repression... 16
Figure 4. Phenotypes of 4-d-old green and red Chinese cabbage with different cultivation conditions. Seedlings and leaves of green and red Chinese cabbages grew on sucrose in the absence (0 mM) or... 37
Figure 5. Total anthocyanin contents in seedlings from green and red Chinese cabbage under various conditions. Data are expressed as the mean values ± standard deviation (SD). Three independent... 38
Figure 6. Transcriptional expression of BrMYBL2.1 and other anthocyanin regulatory and biosynthetic genes under different cultivation conditions in green and red Chinese cabbage. (A) Relative expression... 39
Figure 7. Multiple cDNA sequence alignment of BrPAP1, BrTT8 and BrMYBL2.1 from green and red Chinese cabbage. Conserved nucleotides are shaded by black and partial conservation is shaded by gray. (A) cDNA sequence alignment of BrPAP1. G, green Chinese cabbage; R, red Chinese cabbage. (B)... 44
Figure 8. Sequence polymorphism of amino acid sequence about BrPAP1, BrTT8 and BrMYBL2.1 from green and red Chinese cabbage. Conserved residues are highlighted in black and partial conservation is shaded by gray. Protein sequences were aligned by using GeneDoc. (A) Amino acid sequence... 46
Figure 9. Phylogenetic tree and multiple sequence alignment derived from amino acid sequences of R3-type MYB repressors related in anthocyanin biosynthesis from Chinese cabbage and other plant species. (A) Phylogenetic tree of BrMYBL2.1s and other R3-MYB repressors proteins from other... 47
Figure 10. Physical interaction between BrMYBL2.1-G, BrMYBL2.1-R, BrPAP1 and BrTT8 by yeast two-hybrid assay. The AD and BD are brought in close proximity and the functional transcription factors... 51
Figure 11. In plant assay with Agrobacterium strains harboring BrPAP1, BrTT8, BrMYBL2.1-G and BrMYBL2.1-R. (A) Transient assay with various combinations of BrPAP1, BrTT8, BrMYBL2.1-G and... 55
Figure 12. Relative transcript levels of endogenous anthocyanin biosynthetic genes in tobacco leaves were determined by RT-qPCR, as NtGAPDH was used as a reference gene. Results are the means ± SD... 56
Figure 13. Promoter activation assay with BrPAP1, BrTT8, BrMYBL2.1-G and BrMYBL2.1-R. (A) Effector and reporter constructs were used in the transcriptional activation assay. The effector construct... 58
Figure 14. Construction of CRISPR/Cas9 vector and schematic of the sgRNA-targeting site. pHAtC construction for gene editing of BrMYBL2.1 in Chinese cabbage. Each sgRNAs are marked as... 61
Figure 15. Agrobacterium-mediated transformation in Chinese cabbage (A) Cotyledon-petioles and hypocotyls of Chinese Cabbage were immersed in an infection solution of Agrobacterium. After... 63