Title Page
국문초록
Contents
I. Introduction 11
II. Materials and Methods 15
1. Plant materials 15
2. mRNA isolation and cDNA synthesis 15
3. Suppression subtractive hybridization (SSH) 16
4. Differential screening of cold-inducible genes 18
5. Northern blot analysis. 18
6. Rapid amplification of cDNA ends (RACE) 20
7. Genomic DNA isolation and Southern blot analysis 21
8. Culture media and growth conditions 21
9. Construction of expression plasmids. 22
10. Expression of the slti gene in E. coli 22
11. SDS-polyacrylamide gel electrophoresis (PAGE) 23
12. Purification of recombinant SLTI114 proteins 23
13. Gelatin Zymography 25
14. MMP Enzymatic Activity Assay 26
15. Arabidopsis Transformation 26
III. Results and Discussion 28
1. Cloning, sequencing and phylogenetic analysis of slti114 encoding metalloproteinase 28
2. Genomic organization and expression at transcriptional level of the slti114 cDNA 40
3. SDS-polyacrylamide gel electrophoresis (PAGE) 43
4. Northern analysis cotyledon(soybean) 46
5. Arabidopsis transformation and Selection 48
6. Matrix Metalloproteinase Acrivity assay 50
REFERENCES 52
ABSTRACT 59
Table 1. Sequence homology of the putative autolytic activation... 36
Table 2. Sequence homology of the zinc binding consensus sites... 37
Fig. 1. Diagram of suppression subtractive hybridization (SSH). 19
Fig. 2. Construction of the expression plasmid pET-SLTI114. The... 24
Fig. 3. Nucleotides and the deduced amino acid sequences of... 31
Fig. 4. Schematic presentation of the predicted SLTI114 protein... 32
Fig. 5. Multiple sequence alignment (A) and Pairwise identities (B)... 35
Fig. 6. The hydropathy profile of Matrix Metalloproteinase... 38
Fig. 7. Phylogenetic tree for aligned amino acid sequences of the... 39
Fig. 8. Northern blot analysis or the soybean metalloproteinase... 41
Fig. 9. Southern blot analysis of soybean genomic DNA for the... 42
Fig. 10. Comparsion of the exoressed SLTI114 protein by... 44
Fig. 11. SDS-PAGE analysis of the recombinant proteins produced... 45
Fig. 12. The developmental stage of soybean cotyledon. 47
Fig. 13. Transformation of SLTI114 into Arabidopsis thaliana. 49
Fig. 14. The effect of metalloproteinase activity on differ protein... 51