title page
Contents
List of abbreviations 10
General Introduction : Characterization of fibroin gene in silkworm 11
1. Silkworm 12
2. Fibroin gene and promoter 14
3. Transcriptional regulation of fibroin gene 15
4. Transcription factors of posterior silk gland in Bombyx mori 16
5. Transcriptional regulation 17
5.1. Activation of chromosome structure 17
5.2. Regulation in the transcription level 18
5.2.1. Transcriptional activation (enhancer) 19
5.2.2. Transcriptional repression 20
6. Expression of fibroin gene 22
7. Transgenic silkworms 23
8. Transposon-mediated gene transfer 25
8.1. P element 25
8.2. piggyBac 26
9. The expression of the transgenes 27
10. Analysis for protein binding sites in promoter 28
Chapter 1. Structural characterization of putative transcription factor of silkworm, Bombyx mori 29
Introduction 30
Materials and methods 33
Results 40
Discussion 55
Chapter 2. Construction of transgenic silkworm producing epidermal growth factor in Bombyx mori 59
Introduction 60
Materials and Methods 63
Results 70
Discussion 87
References 89
ABSTRACT 118
국문요약 121
General Introduction 9
Chapter 1 9
Table 1. Expression of silk proteins in posterior silk gland using ESTs data 44
Table 2. Putative transcription factors in posterior silk gland using ESTs data 45
Table 3. Primer sequences for amplification of the using probe 47
Chapter 2 9
Table 1. Injection and transformation of p[FPEGF] vector and study of EGF transgenic animals 74
Table 2. The comparison with normal pupae and transgenic pupae weight 78
General Introduction 7
Chapter 1 7
Fig. 1. cDNA sequence of EST data used as probes. 8-10, 8-83 and peg053 are names in EST data of B. mori. The three ESTs were partially matched with transcription factor of other organisms (shown in round brackets). 46
Fig. 2. Construction of genomic library and plaque hybridization. 48
Fig. 3. Confirmation of probes by PCR with the genomic DNA. 49
Fig. 4. RT-PCR of silkworm according to specific tissues. 50
Fig. 5. Hypothetical structure of counterpart of p8 in B. mori. 51
Fig. 6. Hypothetical structure of counterpart of ASF in B. mori. 52
Fig. 7. Comparisons of hypothetical Asf1 of B. mori. 53
Fig. 8. Comparisons of hypothetical p8 of B. mori. 54
Chapter 2 8
Fig. 1. Construction of expression vector p[FPEGF]. 71
Fig. 2. Analysis of genomic PCR of transgenic silkworm. 75
Fig. 3. Results of comparison with PCR products sequence of transgenic silkworms F2 and EGF gene/SV40 poly (A) signal with used for foreign DNA in microinjection.... 76
Fig. 4. The comparison with normal pupae size and transgenic pupae size. 77
Fig. 5. Result of inverse PCR of genomic DNA in transgenic silkworm. After inverse PCR, we sequenced. The sequence was subjected to BLAST analysis against the nucleotide sequence data present in GenBank. 80
Fig. 6. RT-PCR of transgenic silkworm according to specific tissues. 82
Fig. 7. Dot blot analysis. 84
Fig. 8. ELISA analysis 86