Comparison of Differential Proteins in Normal and Memory-deficient Drosophila melanogaster

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研究生：	李曉芸 Li, Hsiao-Yun
論文名稱：	Comparison of Differential Proteins in Normal and Memory-deficient Drosophila melanogaster 正常果蠅及突變果蠅之差異性蛋白分析
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	71
中文關鍵詞：	果蠅、記憶、二維電泳
外文關鍵詞：	Drosophila melanogaster, Memory, Ephrin receptor, Dek, 2D-DIGE
相關次數：	點閱：3 下載：0
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Learning and memory are not unitary processes. They are extremely complicated and dynamic. Proteins participate in memory formation are tightly regulated by various pathways, and may require protein synthesis and/or post-translational modifications. A memory-related gene (Amnesiac) in Drosophila melanogaster, encodes a neuropeptide which takes part in the memory-related protein kinase A (PKA) activity. Flies with mutated amn possess normal learning and memory behavior within the first 30 minutes of training. However, the process by which that learned can not be retained after 30 minutes of training, indicating these mutants are with deficits in middle-term memory (MTM). Using 2D-DIGE, we compared the protein expression pattern of D. melanogaster wild-type strains (2u, Canton-Special) with memory-deficient mutant (AmnX8). The results of this work provide new insights into understanding of protein expression level during memory formation. Protein spots with different expression levels between wild type and memory-deficient mutants were selected and identified. We identified 30 proteins that are differentially expressed between 2u and AmnX8 in fly brain. Protein 14-3-3 is more abundant in 2u than AmnX8 (9.26-fold). Recent studies indicate that 14-3-3 protein plays an indispensable role in the memory formation. In addition, energy-related proteins, cytoskeleton proteins and iron-related proteins are down-regulated in the mutant strain. Glycerol-3-phosphate dehydrogenase is shown to be post-translationally modified. As a result, glycerol-3-phosphate dehydrogenase plays in learning and memory formation is an alluring theme for future studies.
Eph family is the largest family of receptor tyrosine kinase, which is comprised of 16 receptors and 9 ligands. Eph receptor tyrosine kinase is a single transmembrane protein which is located on post-synaptic neurons, and its molecular weight is 121.5 kDa. Previous studies revealed that after Eph receptor tyrosine kinase interacts with NMDA receptor, calcium ions influx into postsynaptic neuron, causing alterations in synaptic plasticity. Hence, in this study we overexpressed and purified the Drosophila ephrin receptor tyrosine kinase (Dek) in E. coli and generated an antibody that recognizes the carboxyl terminal of Dek. The anti-Dek-C antibody was employed to examine the direct interaction between NMDA and Eph receptors in Drosophila brain.

Contents
Abbreviations ………………………………………………………………………………. iv

I. Comparison of Differential Proteins in Normal and Memory-deficient
Drosophila melanogaster
Chapter I. Introduction
1.1 Learning and Memory…………………………………………….…………...… 1
1.2 Memory related genes…………………………………….………………………. 2
1.3     Animal model…………………………………………….……………………...…. 3
1.4     2D-DIGE…………………………………………………..………………………… 4
1.5    Motivation…………………...……………………………………………………… 4
Chapter II. Materials and Methods
2.1 Drosophila rearing and strains………………..……………………………….… 6
    2.2 Sample preparation…………………………………………………………………. 6
2.3 2D-DIGE…………….…………...…………………………………………………... 7
    2.4 Isoelectric focusing and Two-dimensional electrophoresis............................... 8
2.5 Detection of dye-labelled proteins and gel image analysis…………………..…. 9
    2.6 Gel staining and in-gel digestion………………………………………………..…. 9
    2.7 MALDI-TOF mass spectrometry analysis and database search…………....... 10
Chapter III. Results and Discussion
3.1 Protein expression profiles of 2u and AmnX8 …………………………………… 12
    3.2 Images comparisons and differential protein analysis of 2u and AmnX8……. 12
        3.2.1 14-3-3 protein……………………………………………………...………. 13
        3.2.2 Energy metabolic-related proteins………………………………………. 14
        3.2.3 Iron-related proteins ………………………………………………………. 15
        3.2.4 Cell structure and mobility proteins……………………...………………. 16
3.3 Conclusion…………………………………………………………………………. 17
Figures:
Figure 1. The structures of the cyanine dyes used in 2D-DIGE and their fluorescence emission filters………………………………………… 19
Figure 2. Briefly experiment flow-chart for Proteomes…………………......... 20
Figure 3. Head proteomes of 2u and AmnX8....….……….….….……….…… 21
Figure 4. Image superimposition of 2u and AmnX8 ………..…………….……. 22
Figure 5. The change of differential expression and statistic values between
2u and AmnX8………………...……………………………………….. 25
    Tables:
Table 1. List of protein identified from 2u and AmnX8………………………….. 27
Table 2. List of identified proteins from 2u and AmnX8 grouped in functional categories........................................................................................ 28
II. Expression and Purification of Drosophila Ephrin Receptor Tyrosine Kinase
in E. coli

Chapter I. Introduction
1.1     Eph receptor tyrosine kinase family………………………………………………..… 30
1.2     Eph receptor and ephrin in synapse………………………………………………… 31
1.3     NMDA receptor……………………………………………………………………….… 31
1.4     Motivation………………………………………………………………………..……… 32
Chapter II. Materials and Methods
2.1 Construction of recombinant Dek …………………………………………………….. 33
    2.1.1 Construction of Dek-N into recipient vectors…………...…………………….. 33
    2.1.2 Construction of Dek-C into recipient vectors……………….........….……….. 34
2.2 Expression of constructed proteins………………………………...…………………. 34
    2.2.1 Expression of Dek-N…………………………………………..………………... 34
    2.2.2 Expression of Dek-C………….…………………………...……………………. 35
2.3 Purification of pGEX-6p-3-Dek-C protein………...............………………….………. 36
2.4 bis-Tris SDS-PAGE…………………………………………………………........…….. 37
2.5 Western blot…………………………………………………………………..…………. 37
2.6 Production of the anti-Dek-C antibody……………………………….………...…….. 38
Chapter III. Results and Discussion
3.1 Sequence alignment of Dek with EphB3……….…………………………………….. 39
3.2 Protein topology prediction ………………….………….…………………...………… 39
3.3 Expression test of Dek-N………………………………………………...…………….. 39
3.4 Expression test of Dek-C…………………………………………………………..…... 40
3.5 Culture medium test………………………………………………….…………….…… 41
3.6 Purification of pGEX-6P-3-Dek-C fusion protein.………………………...…….……. 41
3.7 Antibody identification and applications…………………………….………………… 42
3.8 Conclusion……………………………………………………………….....…………… 43
Figures:
Figure 1. The EphB interact with NMDA receptor in the synapses.……….... 44
Figure 2. Sequence alignments of Dek and EphB3…………………………… 45
Figure 3. Topological prediction of Dek………………………………………… 46
Figure 4. Plasmid construction of recombinant His-pET-28a(+)-Dek-N…...... 47
Figure 5. Plasmid construction of recombinant Trx-pET-32a(+)-Dek-N….…. 48
Figure 6. Plasmid construction of recombinant His-pET-28a(+)-Dek-C…...... 49
Figure 7. Plasmid construction of recombinant Trx-pET-32a(+)-Dek-C….…. 50
Figure 8. Purification of Trx-pET-32a(+)-Dek-C with cobalt column……..….. 51
        Figure 9. Plasmid construction of recombinant pGEX-6P-3-Dek-C...……..... 52
Figure 10. Expression and purification of pGEX-6P-3-Dek-C…..…………… 53
Figure 11. Culture medium test for protein production enrichment………. 54
        Figure 12. Purification of pGEX-6P-3-Dek-C protein…….…………………. 55
Figure 13. Identification of co-purified protein with Dek-C.…………………... 60
        Figure 14. Amino acid composition of Dek-C protein…………………………. 61
Figure 15. In vivo assay of anti-Dek-C antibody..……………………………… 62
Tables:
Table 1. Vectors and host cells used for this study………...…………………. 63
Table 2. List of primers used in this study for amplified Dek proteins……...... 64
Table 3. Five culture medium components…………………………………… 65
References………………………………………………………………………………….. 66

                                

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