표제지
ABSTRACT
목차
제Ⅰ절 서론 15
1-1. 본 연구의 목적 23
제Ⅱ절 재료 및 방법 32
1. 식물재료 32
2. sgRNA (single guide RNA) 선정 및 합성 32
3. Ti-plasmid 벡터 구축 33
4. Agrobacterium법을 이용한 형질전환체 육성 34
5. Deep Sequencing 분석에 의한 교정체 확인 35
6. 교정체 후대에서 T-DNA 제거 개체 선발 35
7. 농업형질 조사 36
8. 종자의 외관특성 분석 36
9. 종자 배유내 전분 추출 36
10. 교정체로부터 아밀로스 함량 분석 37
11. 교정체 배유로부터 호화도 측정 37
12. 교정체 배유 결정분석 38
13. RVA(Rapid Visco Analyser) 및 취반·식미 특성 분석 38
14. 교정체의 유리 아미노산 함량 측정 38
15. 탄소 및 질소 함량 분석 39
제Ⅲ절 결과 및 고찰 41
1. OsAAT5, OsATL7 및 OsAAP10 유전자 구조분석 41
2. OsAAT5, OsATL7 및 OsAAP10 유전자 교정식물체 육성 51
3. OsAAT5, OsATL7 및 OsAAP10 유전자 교정체 확인 56
4. OsAAT5, OsATL7 및 OsAAP10 유전자 교정체의 특성분석 66
1) 교정체 후대에서 null 식물체 선발 및 T₂ 종자 육성 66
2) 교정체 후대에서 농업형질 조사 70
3) 교정체 후대에서 종자 외관과 전분 과립 분석 73
4) 교정체 후대에서 아밀로스 함량 분석 75
5) 교정체 후대에서 배유 호화도 분석 77
6) 교정체 후대에서 전분 결정 구조 분석 79
7) 교정체 후대에서 식미 특성 분석 81
8) OsAAP10 교정체 후대에서 아미노산 함량 분석 86
9) OsAAP10 교정체 후대에서 C/N 비율 분석 89
제Ⅳ절 결론 91
제Ⅴ절 참고문헌 94
국문요약 103
Table 1. Operating conditions of amino acid auto-analyzer 40
Table 2. Selected sgRNA of OsAAT5, OsATL7, OsAAP10 in rice genome using CRISPR RGEN tools. 53
Table 3. Frequency of transgenic plants by using CRISPR/Cas9 vector in rice. 58
Table 4. Count of mutant genotype and mutation type at the target site in mutant plants. 62
Table 5. Chi-square analysis for resistant and susceptible strains, estimated with the bar-strip test from T₁ generation of OsAAT5, OsATL7, OsAAP10 edited plants. 69
Table 6. Agronomic traits of OsAAT, OsATL7, OsAAP10 lines in T₃ generation. 71
Table 7. Rapid Visco Analyzer(RVA) and eating and cooking quality(ECQ) analysis of the OsAAT10 mutant lines. 82
Figure 1. Carbon source partitioning and amino acid synthesis in plant cells Carbon source partitioning with glucose as the carbon source is shown. Basic carbon energy metabolism is defined as pathway 1,... 24
Figure 2. Ripening process of rice seeds. The graining process of rice seeds and the storage process of stored substances are shown. Each stage of 0-7DAF is shown as a schematic diagram of the development... 25
Figure 3. Organelles localized in the endosperm tissue of fully ripened seeds. The endosperm tissue is composed of starchy endosperm tissue surrounded by aleurone tissue. The image is a transmission... 26
Figure 4. Nitrogen response in plants. The nitrogen response in plants is shown schematically. Plants use ammonium ions and nitrate ions contained in the soil as nitrogen sources. Inorganic nitrogen... 27
Figure 5. Rice seed storage proteins. (A) Rice seed storage proteins were fractionated by SDS-PAGE. (B) Prolamin distribution revealed by immunofluorescence. It has a layered structure with 10 kDa... 28
Figure 6. Ingredient composition of rice and its effect on taste. The food ingredients contained in 100 g of brown rice and the sensory evaluation items of the taste given by them are shown. 29
Figure 7. Translocation pathway of photosynthetic products into the endosperm and the sequence of starch synthesis. At the early stage of grain filling, sucrose flows toward the center... 30
Figure 8. Schematic diagram of starch synthesis in seeds. 31
Figure 9. Schematic structure of OsAAT5 gene. Genomic DNA region and structure of OsAAT5 gene from chromosome 1 of Oryza sativa and cDNA sequences. OsAAT5 has a genome size of 3.62kb and... 42
Figure 10. Schematic structure of OsATL7 gene. Genomic DNA region and structure of OsATL7 gene from chromosome 1 of Oryza sativa and cDNA sequences. OsATL7 has a genome size of 6.38kb and... 43
Figure 11. Schematic structure of OsAAP10 gene. Genomic DNA region and structure of OsAAP10 gene from chromosome 2 of Oryza sativa and cDNA sequences. OsAAP10 has a genome size of 7.41kb and... 44
Figure 12. Nucleotide and deduced amino acid sequences of a OsAAT5 gene encoding amino acid transporter in rice (Oryza sativa L.). 45
Figure 13. Nucleotide and deduced amino acid sequences of a OsATL7 gene encoding amino acid transporter in rice (Oryza sativa L.). 46
Figure 14. Nucleotide and deduced amino acid sequences of a OsAAP10 gene encoding amino acid transporter in rice (Oryza sativa L.). 47
Figure 15. Alignment and deduced amino acid sequences of the coding regions of OsAAT5 genes (AP014957.1; NCBI data, AAT5; Isolated in this study). Genebank accession numbers are Oryza sativa,... 48
Figure 16. Alignment and deduced amino acid sequences of the coding regions of OsATL7 genes (XM_015771077.2; NCBI data, ATL7; Isolated in this study). Genebank accession numbers are Oryza... 49
Figure 17. Alignment and deduced amino acid sequences of the coding regions of OsAAP10 genes (XM_026022869.1; NCBI data, AAP10; Isolated in this study). Genebank accession numbers are Oryza... 50
Figure 18. Ti-plasmid vector (pBOsC) construction of sgRNA region for CRISPR/Cas9-mediated mutagenesis in the starch biosynthesis genes in rice. LB; left border; RB, right border; OsU3-P; Oryza... 54
Figure 19. Agrobacterium-mediated transformation procedures. a, callus formation; b, selection of callus; c, Agrobacterium-mediated transformation; d-e, selection of embryogenic callus; f, green spot... 55
Figure 20. PCR analysis of bar gene and nos terminator region from pBOsC::OsAAT5 regeneration plants. (A) pBOsC::OsAAT5_sg1 regeneration plants. (B) pBOsC::OsAAT5_sg2 regeneration plants. M,... 59
Figure 21. PCR analysis of bar gene and nos terminator region from pBOsC::OsATL7 regeneration plants. (A) pBOsC::OsATL7_sg1 regeneration plants. (B) pBOsC::OsATL7_sg2 regeneration plants. M,... 60
Figure 22. PCR analysis of bar gene and nos terminator region from pBOsC::OsAAP10 regeneration plants. (A) pBOsC:: OsAAP10_sg1 regeneration plants. (B) pBOsC:: OsAAP10_sg2 regeneration plants.... 61
Figure 23. NGS(Next Generation Sequencing) analysis result of OsAAT5_sg1, OsAAT5_sg2. Inserted sequences in mutant are indicated by red, and dash(-) signify deletion sequences. 63
Figure 24. NGS(Next Generation Sequencing) analysis result of OsATL7_sg1, OsATL7_sg2. Inserted sequences in mutant are indicated by red, and dash(-) signify deletion sequences. 64
Figure 25. NGS(Next Generation Sequencing) analysis result of OsAAP10_sg2. Inserted sequences in mutant are indicated by red, and dash(-) signify deletion sequences. 65
Figure 26. T₁ plants growing in experimental greenhouse of Chungbuk National University, daytime temperature(33-35℃)/night temperature(above 20℃). 67
Figure 27. The Bar gene screening with Basta treatment on leaf tip and Bar-strip analysis in T₁ generation. (A) Selection of OsAAT5, OsATL7, OsAAP10 transgenic T₀ plants after 40 ppm basta... 68
Figure 28. Shape comparison for wild type and OsAAT5, OsATL7, OsAAP10 plants. 72
Figure 29. CRISPR/Cas9 mediated editing of OsAAT5, OsATL7, OsAAP10 gene. (A) Phenotypes of seeds about brown rice and cross sections. (B) Scanning electron microscopy analysis of starch granule morphology. 74
Figure 30. Physicochemical traits of starch of OsAAT5, OsATL7, OsAAP10 mutants and wild-type. Amylose content of polished grains in rice. 76
Figure 31. Physicochemical traits of starch of OsAAT5, OsATL7, OsAAP10 mutants and wild-type. I₂-KI absorption spectroscopic analysis of the gelatinized supernatant fractions from rice flours in 4 M... 78
Figure 32. Physicochemical traits of starch of OsAAT5, OsATL7, OsAAP10 mutants and wild-type. X-ray diffraction (XRD) patterns of endosperm starch granules. 80
Figure 33. Starch RVA profiles of the OsAAT5 mutants. There were smiliar in mutant and WT hardness of grains. There were lower in mutant line than in WT in the adhesiveness of grains. 83
Figure 34. Starch RVA profiles of the OsATL7 mutants. There were smiliar in mutant and WT hardness of grains. There were lower in mutant line than in WT in the adhesiveness of grains in rice. 84
Figure 35. Starch RVA profiles of the OsAAP10 mutants. There were smiliar in mutant and WT hardness of grains. There were lower in mutant line than in WT in the adhesiveness of grains in rice. 85
Figure 36. Amino acid content in grains at maturity of rice. For mutants, there were 20% lower in mutant line than in WT in the contents of all detected amino acids in grains. 87
Figure 37. Amino acid content in grains at maturity of rice. Asp, Glu, Ala, Gly, Pro, Ser, Thr, Asn and Arg represent aspartate, glutamate, alanine, glycine, proline, serine, threonine, asparagine and arginine, respectively. 88
Figure 38. Carbon and Nitrogen content analysis about OsAAP10 mutant line. C/N ratio analysis using Carbon and Nitrogen content in T₂ generation. 90