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研究生:阮慶隆
研究生(外文):NGUYEN THANH LONG
論文名稱(外文):Analyzing The Proteins From The Bacteria
指導教授:劉昭麟劉昭麟引用關係
指導教授(外文):LIU, CHAO-LIN
口試委員:劉昭麟丁慧如陳錫金
口試委員(外文):LIU, CHAO-LINTING, HUEI-JUCHEN, CHIN-LUNG
口試日期:2022-08-03
學位類別:碩士
校院名稱:明志科技大學
系所名稱:化學工程系生化工程碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:英文
論文頁數:73
外文關鍵詞:Ralstonia solanacearumLelliottia amnigenaProtein2D-gel
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Bacterial wilt is one of the most devastating agricultural diseases in the world, most commonly affecting tomatoes. Four strains were identified based on colony morphology on TTC medium and PCR products homologous to primers pairs 759-760 and 16S-23S rRNA.
Three of them were identified as R. solanacearum and one of them was L. amnigena. L. amnigena. Pathogenicity in tomatoes proves that L. amnigena has the ability to cause bacterial wilt. Therefore, it is necessary to analyze the basic protein system of these two species, the main cause of bacterial wilt in tomatoes, to find ways to prevent wilt disease in future studies. In this study, we had tested the pathogenicity on tomato plants of four bacterial strains and preliminarily analyzed their proteins by 2D-gel.
Strains PSS4 and 581 were death rates were 30% and 33% after 30 days of infection. Strains S2 and L2R showed symptoms of green wilt after 15 days, with death at the rate of 10% and 23.3% after 30 days. The total protein extracted from strain S2 was completely higher than that of other strains. There is a relative similarity of protein between two strains PSS4 and 581.

Recommendation Letter from the Thesis Advisor i
Thesis Oral Defense Committee Certification ii
ACKNOWLEDGMENTS iii
ABSTRACT iv
TABLE OF CONTENTS v
ABBREVIATION LIST viii
LIST OF FIGURES x
LIST OF TABLES xii
CHAPTER 1: INTRODUCTION 1
1.1. Background 1
1.2. Objectives 2
1.3. Scope of the study 2
CHAPTER 2: LITERATURE REVIEW 3
2.1. Distribution and host range 3
2.2. Nomination 3
2.3. Characteristics of R. solanacearum 4
2.3.1. Morphological characteristics of R. solanacearum 4
2.3.2. Biochemical properties of R. solanacearum bacteria 5
2.3.3. Mode of survival, invasion and spread of R. solanacearum 6
2.3.4. Classification 7
2.3.5. Proteomics of R. solanacearum 9
2.3.6. Three types colonies of R. solanacearum 11
2.3.7. Diagnosis of wilt disease 11
2.4. PAGEs (polyacrylamide gel electrophoresis) 12
2.4.1. SDS-PAGE 12
2.4.2. Two-Dimensional SDS-PAGE 12
2.4.3. Native PAGE 12
CHAPTER 3: MATERIALS AND METHODS 14
3.1. Materials and chemicals 14
3.1.1. Materials 14
3.1.2. Chemicals 14
3.1.3. Instruments 15
3.1.4. Chemical preparation 15
3.2. Research methods 17
3.2.1. Bacteria isolation and cultivation 17
3.2.2. Bacteria identification 17
3.2.3. Investigation of possible pathogenicity 21
3.2.4. Protein extraction from bacteria 21
3.2.5. Protein quantification 21
3.2.6. Protein separation with 2-D SDS-PAGE 22
CHAPTER 4: RESULTS AND DISCUSSION 24
4.1. Bacteria Isolation 24
4.2. Molecular identification 24
4.2.1. DNA extraction 24
4.2.2. Molecular analysis 24
4.2.3. Bacteria identification 25
4.3. Pathogenicity test on tomato 26
4.3.1. Cell counts number bacteria 26
4.3.2. Pathogenicity test on tomato 27
4.4. Protein quantification 27
4.4.1 BSA standard curve 27
4.4.2 Sample quantification 27
4.5. Two-dimension electrophoresis 27
CHAPTER 5: CONCLUSION 28
REFERENCES 59

LIST OF FIGURES
Figure 1: Vector map 29
Figure 2: Virulent (mucoid, pink-colored) individual colonies of R. solanaceraum (PSS4) on TTC agar growth medium 30
Figure 3: Virulent (mucoid, pink-colored) individual colonies of R. solanaceraum (581) on TTC agar growth medium 31
Figure 4: Lelliottia amnigena (S2) on TTC agar growth medium 32
Figure 5: R. solanaceraum strain QK-2 (L2R) on TTC agar growth medium 33
Figure 6: Three morphological colony forms of R. solanacearum developed on TTC medium after 3 days growth at 30 °C.. 34
Figure 7: PCR amplified with primer pairs 759-760 in various concentrations 35
Figure 8: DNA fragments amplified by primer pairs 759-760 on the genomic DNA extracted from bacteria with PCR. The DNA fragments were analyzed on 2% agarose 36
Figure 9: DNA fragments amplified by primer pairs 16S-23S on the genomic DNA extracted from bacteria with PCR. The DNA fragment were analyzed on 2% agarose 37
Figure 10: The relationship between the S2 cell number and the absorbance at 600 nm of the cell cultured 38
Figure 11: The relationship between the PSS4 cell number and the absorbance at 600 nm of the cell cultured. 39
Figure 12: The relationship between the 581 cell number and the absorbance at 600 nm of the cell cultured 40
Figure 13: The standard curve of protein quantification with the BSA as the standard. 41
Figure 14: 2D analysis of the protein extract from PSS4 with pH 3–11 NL using CBB staining. 42
Figure 15: 2D analysis of the protein extract from 581 with pH 3–11 NL using silver staining. 43
Figure 16: 2D analysis of the protein extract from S2 with pH 3–11 NL using silver staining. 44
Figure 17: 2D analysis of the protein extract from L2R with pH 3–11 NL using CBB staining. 45

LIST OF TABLES
Table 1: Ralstonia solanacearum current taxonomic state 46
Table 2: Classification of R. solanacearum strains into biovars based on oxidation of three disaccharides and three hexose alcohols 47
Table 3: Phylotypes of the Ralstonia solanacearum species complex 48
Table 4: Classification of R. solanacearum strains into species 49
Table 5: DNA concentration of bacteria 50
Table 6: DNA concentration of bacteria after recovery 51
Table 7: Components ligation with Topo pCRⅡ vector 52
Table 8: The colony PCR thermal cycle 53
Table 9: Primers for PCR reaction 54
Table 10: Identification results 55
Table 11: The results of the survey on the pathogenicity of bacteria on tomato plants 56
Table 12: The optical density of variant BSA concentration at A660 nm 57
Table 13: The protein concentration of sample (mg/mL) 58



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