표제지
ABSTRACT
목차
Nomenclature 12
제1장 서론 14
제2장 재료 및 방법 21
1) 식물재료 및 생장조건 21
2) Single guide RNA(sgRNA) 선정 22
3) sgRNA 합성 및 Ti-plasmid 벡터 구축 23
4) Agrobacterium을 이용한 재분화체 생산 및 T-DNA 도입 확인 25
5) 유전자 변이체 선발 및 후대육성 26
6) T₁ 변이 계통에서 null 개체 선발 27
7) 생화학적 분석 28
(1) Carotenoid 함량측정 28
(2) GABA 및 free amino acid 함량측정 28
8) RNA sequencing 및 data 분석 29
9) RT-qPCR 분석 30
제3장 결과 및 고찰 31
1) 유전자 구조분석 31
2) Single guide RNA (sgRNA)의 선정 38
3) CRISPR/Cas9 발현 Ti-plasmid 벡터 구축 확인 40
4) 형질전환체 육성 및 선발 44
5) 유전자 변이체 선발 및 분석 47
6) 형질전환 T₁ 계통 육성 및 null 선발 51
7) HPLC system을 이용한 대사산물 측정 57
(1) Carotenoid 함량 분석 57
(2) GABA 및 Total-amino acid 함량 59
8) RNA-sequencing분석을 통한 전사체 해석 61
9) 유전자 발현 분석 70
제4장 결론 76
참고문헌 79
Supplementary data 86
국문요약 119
Table 1. Mutation frequencies at on–target and potential off–target sites among selected sgRNA target of the carotenoid biosynthesis pathway and GABA shunt genes in tomato genome using RGEN tools. 39
Table 2. Ratios of mutant genotype and mutation type at the target site in T₀ mutant plants 48
Table 3. Chi–square test for null selection. 53
Table 4. Phenotype characterization in tomato organ. 56
Table 5. Raw data, pre-processing data and Mapping statistics of RNA–seq data from six RNA–sequencing analysis. 63
Table 6. Top five GO of cluster in S1 #1–11 vs WT_SGR1and S1 #2–12 vs WT_SGR1. 68
Table 7. Top five GO of cluster in G2 #1–1 vs WT_GAD and G3 #2–4 vs WT_GAD. 69
Figure 1. Overview of carotenoid biosynthesis pathway in tomato plant. Key enzymes involved in carotenoid biosynthesis include geranylgerany1 pyrophosphate(GGPP), phytoene synthase 1(PSY1),... 19
Figure 2. GABA shunt pathway in higher plant. Key enzymes involved in GABA shunt include glutamate decarboxylase(GAD), GABA transaminase(GABA–T), Succinate Semialde Hyde Dehydrogenase(SSADH). 20
Figure 3. Nucleotide and deduced amino acid sequence of SlSGR1 full length cDNA isolated from Solanum lycopersicum. L sgRNA sequence is indicated blure color. 32
Figure 4. Nucleotide and deduced amino acid sequence of SlGAD2 full length cDNA isolated from Solanum lycopersicum. L sgRNA sequence is indicated blure color. 33
Figure 5. Nucleotide and deduced amino acid sequence of SlGAD3 full length cDNA isolated from Solanum lycopersicum. L sgRNA sequence is indicated blure color and underline. 34
Figure 6. Alignment and deduced amino acid sequences of the coding regions of SGR1 protein[Solanum lycopersicum, NM_001247794(Tomato); Solanum pennellii, XM_015229006(wild tomato);... 35
Figure 7. Alignment and deduced amino acid sequences of the coding regions of GAD2 protein[Solanum lycopersicum, NM_001246893 (Tomato); Solanum pennellii, XM_015202620(wild tomato);... 36
Figure 8. Alignment and deduced amino acid sequences of the coding regions of SGR1 protein[Solanum lycopersicum, NM_001246898 (Tomato); Solanum pennellii, XM_015216838 (wild tomato);... 37
Figure 9. Digestion of Xho I restriction enzyme from PCR products of sgRNA region. M, molecular marker; Mock, Mock control as negative control pKAtC vector plasmid DNA without sgRNA; Xho I... 41
Figure 10. Alignment analysis of sgRNA sequences of carotenoid synthesis pathway and GABA shunt genes. Identical, sgRNA regions are indicated by asterisks(*) 42
Figure 11. Ti-plasmid vector (pKAtC) construction of sgRNA region for CRISPR/Cas9-mediated mutagenesis in the SlSGR1, SlGAD2 and SlGAD3 genes in tomato. LB, left border; RB, right border;... 43
Figure 12. Development of transgenic tomato. (A) Introduction of pKAtC vector through Agrobacterium–mediated transformation in tomato plants. (B) Schematic of tomato plant transformation... 45
Figure 13. PCR analysis of Kanamycine–R region. M, molecular marker; P.C, pKAtC Vector as control; N.C, Negative control; Each level generated from the DNA template of independent T₀ transgenic. 46
Figure 14. Targeted Next Generation Sequencing (NGS) analysis of CRISPR/Cas9–induced SlSGR1 gene modification in tomato. Deletion and insertion sequences indicated dash and red letters,... 49
Figure 15. Targeted Next Generation Sequencing (NGS) analysis of CRISPR/Cas9–induced SlGAD gene modification in tomato and amino acid sequences of targeted regions. Deletion and insertion sequences indicated dash and red letters, respectively. The underlined green letters indicate the C–... 50
Figure 16. Null selection by PCR analysis using kanamycin regions amplification. No band indicates that T–DAN has been removed. 52
Figure 17. Resequencing analysis of gene edited plants. Mapping of genome sequences of T₁ null lins. Light blue highlights indicate the annotated vector regions. The solid red line marks... 54
Figure 18. Phenoype of T₁ transgenic plants and fruits. 55
Figure 19. Determination of carotenoid levels in S1 #1–11, S1 #2–12 and wild-type (WT) plant fruits at fruit ripening stage. 58
Figure 20. Determination of GABA and Amino acid levels in G2 #1–1, G3 #2–4 and wild type (WT) plant fruits at fruit ripening stage. 60
Figure 21. MA plot of differentially expressed genes identified in T₁ null lines vs WT. Data represent individual gene responses plotted as log₂ fold-change versus Means of count fold-change〉2 (p–values... 64
Figure 22. Comparison the number of differentially expressed genes (DEGs) in T₁ null lines and WT. (A) Venn diagrams display the number of up or down regulated genes. (B) Graph display the number of... 65
Figure 23. Heat map and line plot of cluster in S1 #1–11 vs WT_SGR1and S1 #2–12 vs WT_SGR1. (A) Heat map showing comparison combination data of S1 #1–11 vs WT_SGR1and S1 #2–12 vs... 66
Figure 24. Heat map and line plot of cluster in G2 #1–1 vs WT_GAD and G3 #2–4 vs WT_GAD. (A) Heat map showing comparison combination data of G2 #1–1 vs WT_GAD and G3 #2–4 vs WT_GAD.... 67
Figure 25. Expression levels of key genes in the carotenoid biosynthetic pathway in tomato fruit at the Br stage. 71
Figure 26. Expression levels of key genes involved in the carotenoid biosynthetic pathway at each ripening stage of tomato fruit. The genes shown are only involved in lycopene synthesis. 72
Figure 27. Expression levels of key genes involved in the carotenoid biosynthetic pathway at each ripening stage of tomato fruit. The genes shown are only involved in lycopene degradation. 73
Figure 28. Expression levels of key genes in the GABA shunt pathway in tomato fruit at the Br stage. 74
Figure 29. Expression levels of key genes involved in the GABA shunt pathway at each ripening stage of tomato fruit. 75