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Abstract in Korean
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List of Abbreviations 10
I. Introduction 16
II. Materials and Methods 24
II-1. Plant and Bacterial strains 24
II-2. Plasmids 24
II-3. Plant growth and stress treatments 24
II-4. Isolation of total RNA from plant 25
II-5. Isolation of poly(A)+ RNA from total RNA 26
II-6. cDNA subtraction 27
II-7. Generation of full-length cDNA 30
II-8. Northern blot analysis 30
II-9. Polymerase chain reaction (PCR) amplification 31
II-10. Reverse transcription 31
II-11. Sequence analysis 32
II-12. Yeast two-hybrid assay 32
III. Results and Discussion 34
III-1. Screening of Dehydration Stress-Inducible Genes in Brassica napus 34
III-1-1. cDNA subtraction 35
III-1-2. Identification of dehydration inducible genes 37
III-1-3. Partial cDNA sequence of dehydration inducible clones 39
III-1-4. Full-length cDNA cloning 48
III-1-5. Sequence analyses of the Bnrd7(Bnp5cs1 and Bnp5cs2), Bnrd114 and Bnrd116 51
III-1-5-1. Sequence analysis of Bnrd7 (Bnp5cs1 and Bnp5cs2) 51
III-1-5-2. Functional complementation of λ-GK and λ-GPR deficient E. coli with Bnp5cs1 and Bnp5cs2 57
III-1-5-3. Sequence analysis of Bnrd114 62
III-1-5-4. Sequence analysis of Bnrd116 65
III-2. Isolation and Characterization of a Dehydration Stress-Inducible Protein Kinase Gene in Brassica napus 68
III-2-1. Sequence analysis of Bnrd197 74
III-2-2. Bnrd197 transcription level under water stree and ABA treatment 81
III-2-2-1. Increased Bnrd197 mRNA levels under dehydration-stressed leaves 81
III-2-2-2. Increased Bnrd197 mRNA levels in salt-stressed leaves 81
III-2-2-3. Increased Bnrd197 mRNA levels in leaves of cold-stressed plants 82
III-2-2-4. Increased Bnrd197 mRNA levels in leaves of cold-stressed plants 82
III-2-2-5. Increased Bnrd197 mRNA levels in leaves exogenously treated with ABA 82
III-2-2-6. Increased Bnrd197 mRNA levels in leaves in osmotic-stressed leaves 89
III-2-3. Effects of light, nutrient and various phytohormones on expression of Bnrd197 89
III-2-3-1. Transcription level of the Bnrd197 gene by exogenous treatment of various phytohormenes 89
III-2-3-2. Effects of light and nutrient on Bnrd197 transcription level 92
III-2-4. The BnRD197 protein interacts specifically with AtCBL1 and AtCBL4/SoS3 92
IV. References 101
V. Summary 130
감사의 글 134
Figure 1. Overview of isolation and characterization of dehydration stress-inducible genes 23
Figure 2. Schematic diagram of cDNA subtraction based on selective amplification of differentially expresed sequences 29
Figure 3. Subtracted partial cDNA clone pattern 36
Figure 4. Northern hybridization analysis using each subtracted partial cDNA probe 38
Figure 5. The partial cDNA sequence of dehydration stress induced clones 43
Figure 6. Agarose gel showing the products 5' RACE(A) and 3' RACE(B) using each gene specific primers 49
Figure 7. Agarose gel showing the products of 5' and 3' RACE using Bnrd197 gene specific primer 50
Figure 8. Nucleotide sequence of Bnp5cs1 (GenBank accession number. AF314811) and its deduced amino acid sequence 53
Figure 9. Nucleotide sequence of Bnp5cs2 (GenBank accession number. AF314812) and its dduced amino acid sequence 55
Figure 10. Multiple alignment of predicted BnP5CS1 and BnP5CS2 protein sequences with homologous sequence of Arabidopsis and moth bean P5CS proteins 56
Figure 11. Complementation of a proBA mutation by Bnp5cs1 and Bnp5cs2 gene 58
Figure 12. Pathways of proline biosynthesis and degradation in plants 60
Figure 13. Nucleotide sequence of Bnrd114 (GenBank accession number. AF101038) and its deduced amino acid sequence 63
Figure 14. Comparison of deduced amino acid sequence of BnRD114 and other lipid transfer proteins 64
Figure 15. Nucleotide sequence of Bnrd116 (GenBank accession number AF314810) and its deduced amino acid sequence 67
Figure 16. Bnrd197 (GenBank accession number, AF319169( encodes a putative serine/threonine protein kinase 77
Figure 17. The alignment of the deduced amino acid sequence of BnRD197 and members of the SNF1-related protein kinase subfamily 3 78
Figure 18. Amino acid sequence alignment of BnRD197 and other Arabidopsis CIPKs 79
Figure 19. Phylogenic tree (A) for subfamily proteins of SnRKs and CIPK proteins and phylogenic tree (B) for the C-terminal region of these proteins 80
Figure 20. Expression of Bnrd197 gene in response to dehydration stress 83
Figure 21. Expression of the Bnrd197 gene in response to salinity stress 84
Figure 22. Expression of the Bnrd197 gene in response to cold stress 85
Figure 23. Expression of the Bnrd197 gene in response to heat stress 86
Figure 24. Expression of the Bnrd197 gene in response to abscisic acid 87
Figure 25. Expression of the Bnrd197 gene in response to osmotic stress 88
Figure 26. Expression of the Bnrd197 gene in response to various phytohormones 90
Figure 27. Expression of the Bnrd197 gene in response to light and nutrient 91
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