臺灣博碩士論文加值系統

香蕉萎縮病係由香蕉萎縮病毒(banana bunchy top virus, BBTV)所引起，在香蕉植株上造成葉片邊緣黃化、葉脈透化、中肋及葉柄處產生濃綠色條斑、嚴重者甚至不結果等病徵。目前研究BBTV之基因體組成已發現至少六條DNA components，分別為DNA1 ~ 6，在某些BBTV分離株上甚可分離到額外的DNA components (additional Rep)，根據病徵及利用PCR三對引子對(C1、S、SR)之檢測結果可將BBTV分成五種系統(type I ~ V)。然而在台灣，不同BBTV系統之完整基因體組成尚未被報導。本研究針對BBTV不同系統之分離株，利用保守性及專一性引子對，對BBTV不同系統分離株之香蕉總核酸進行PCR反應分析，增幅出可能具有的基因體，建立出PCR library，再利用restriction fragment length polymorphism (RFLP)對PCR library內之選殖株進行分析、選殖並定序來自不同RFLP群之DNA components，從type I-1與II-1兩分離株當中找到六個DNA components，並發現兩者之間除DNA2a外，各DNA components全長序列、major common region (CR-M)與stem-loop common region (CR-SL)相似度極高，主要差異在SR引子對產物(即additional Rep)之有無，進一步分析其中所含有之additional Rep種類並加以分群，同一type不同additional Rep群之間病徵並無顯著差異，對於影響type I與II之間病徵強弱之差異仍需進一步研究；從type III分離株當中只找到兩個序列特異性之DNA components (L1與L2)，且無發現任何已知additional Rep的存在，其中L1為本研究中新發現之DNA component；type IV分離株只找到DNA1 ~ 5，並發現部分DNA1 與DNA3 ~ 5之CR-SL序列具有缺失，而DNA2則具有特異性CR-SL序列，並從type IV分離株發現一新的additional Rep (Ly1)。

目次
、前 研究................................................................................................................1
( ) 香蕉萎縮毒病簡介................................................................................1
( ) BBTV基因體之研究..............................................................................2
( ) BBTV病毒系統之研究..........................................................................9
、材料與方法............................................................................................................9
( ) 植物材料..............................................................................................11
( ) 總核酸抽取..........................................................................................11
( ) 熱循環反應..........................................................................................11
1. C1及SR引子對........................................................................11
2. S引子對....................................................................................12
3. Y、S1、S2、S3之各專 性引子對.......................................12
4. 其他各DNA專 性引子對…................................................12
( ) 核酸之選殖..........................................................................................12
( ) 製備大腸桿菌Top10F’或XL1-Blue勝任細胞...................................12
( ) 大腸桿菌Top10F’或XL1-Blue勝任細胞之轉形...............................13
( ) 菌落熱循環反應..................................................................................13
( ) 質體少量製備......................................................................................13
( ) DNA定序.............................................................................................14
( ) DNA序列分析.....................................................................................14
、結果......................................................................................................................15
( ) BBTV各系統主要基因體之選殖與分析方法....................................15
( ) BBTV各系統分離��之PCR library建立與分析...............................15
1. BBTV type I-1.........................................................................15
(1) BBTV type I-1之C1 library與內含基因體之分析....15
(2) BBTV type I-1之S library與內含基因體之分析......16
(3) BBTV type I-1之SR library與內含基因體之分析....16
(4) BBTV type I-1之DNA2, 4, 6之選殖與定序..............17
BBTV type I-1之DNA2 library與內含基因體之分析..17
BBTV type I-1之DNA4 library與內含基因體之分析..........17
 BBTV type I-1之DNA6 library與內含基因體之分析..17
2. BBTV type II-1........................................................................18
(1) BBTV type II-1之C1 library與內含基因體之分析..18
(2) BBTV type II-1之S library與內含基因體之分析.....18
(3) BBTV type II-1之DNA2, 4, 6之選殖與定序............19
BBTV type II-1之DNA2 library與內含基因體之分析....
...........................................................................................19
BBTV type II-1之DNA4 library與內含基因體之分析....
...........................................................................................19
 BBTV type II-1之DNA6 library與內含基因體之分析.....
............................................................................................19
3. BBTV type III..........................................................................20
(1) BBTV type III之S library與內含基因體之分析.......20
(2) BBTV type III之DNA1 ~ 6之選殖與定序................20
4. BBTV type IV..........................................................................21
(1) BBTV type IV之C1 library與內含基因體之分析....21
(2) BBTV type IV之SR library與內含基因體之分析....21
(3) BBTV type IV DNA1 ~ 6之選殖與定序...................22
BBTV type IV之DNA1 library與內含基因體之分析..22
BBTV type IV之DNA2 library與內含基因體之分析..22
 BBTV type IV之DNA3 library與內含基因體之分析..23
 BBTV type IV之DNA4 library與內含基因體之分析..23
 BBTV type IV之DNA5 library與內含基因體之分析..23
 BBTV type IV之DNA6 library與內含基因體之分析..23
( ) BBTV各系統分離��主要基因體之序列分析........................................25
1. BBTV type I-1.........................................................................25
(1) BBTV type I-1-1序列分析......................................................25
(2) BBTV type I-1-2序列分析..........................................25
(3) BBTV type I-1-3序列分析..........................................25
(4) BBTV type I-1-4序列分析..........................................25
(5) BBTV type I-1-5序列分析..........................................26
(6) BBTV type I-1-6序列分析..........................................26
(7) BBTV type I-1-1 ~ 6之CR-SL區域比較....................26
2. BBTV type II-1........................................................................26
(1) BBTV type II-1-1序列分析.........................................26
(2) BBTV type II-1-2序列分析.........................................27
(3) BBTV type II-1-3序列分析.........................................27
(4) BBTV type II-1-4序列分析.........................................27
(5) BBTV type II-1-5序列分析.........................................27
(6) BBTV type II-1-6序列分析.........................................28
(7) BBTV type II-1-1 ~ 6之CR-SL區域比較...................28
3. BBTV type III..........................................................................28
(1) BBTV type III之L1序列分析.....................................28
(2) BBTV type III之L2序列分析.....................................28
(3) BBTV type III之DNA1 ~ 6之序列分析....................29
(4) BBTV type III L1與L2之CR-SL區域比較.................29
4. BBTV type IV..........................................................................29
(1) BBTV type IV-1之序列分析......................................29
(2) BBTV type IV-2a與IV-2b之序列分析.......................29
(3) BBTV type IV-3之序列分析......................................30
(4) BBTV type IV-4之序列分析......................................30
(5) BBTV type IV-5之序列分析......................................30
(6) BBTV type IV之DNA6序列分析..............................30
(7) BBTV type IV-1 ~ 6之CR-SL區域比較.....................31
( ) BBTV各系統分離��主要基因體之序列比較與親緣演化分析........31
1. BBTV type I-1 ~ IV基因體之全長序列比較與親緣演化分析.
..................................................................................................31
(1) BBTV type I-1 ~ IV基因體之全長核 酸序列比較.31
(2) BBTV type I-1 ~ IV基因體轉譯之胺基酸序列比較.31
2. BBTV type I-1 ~ IV基因體CR-SL區域之序列比較與親緣演
化分析.......................................................................................32
3. BBTV type I-1 ~ IV CR-M區域之序列比對與演化樹分析.32
4. 與各國BBTV分離��之DNA1 ~ 6序列比較與親緣演化分析..
..................................................................................................32
(1) DNA1之序列比較與親緣演化分析...........................33
(2) DNA2之序列比較與親緣演化分析...........................33
(3) DNA3之序列比較與親緣演化分析...........................33
(4) DNA4之序列比較與親緣演化分析...........................34
(5) DNA5之序列比較與親緣演化分析...........................34
(6) DNA6之序列比較與親緣演化分析...........................34
( ) BBTV各系統分離��additional Rep基因體之選殖與分析方法......34
( ) BBTV各系統分離��additional Rep之PCR library建立與分析.....35
1. BBTV type I-1.........................................................................35
(1) BBTV type I-1之Y library與內含基因體之分析.....35
(2) BBTV type I-1之S1 library與內含基因體之分析....35
(3) BBTV type I-1之S2 library與內含基因體之分析....36
(4) BBTV type I-1之S3 library與內含基因體之分析....36
(5) BBTV其他type I分離��之Y、S1、S2及S3 library與
內含基因體之分析.......................................................36
2. BBTV type II-1........................................................................37
(1) BBTV type II-1之Y library與內含基因體之分析....37
(2) BBTV type II-1之S1、S2及S3 library與內含基因體
之分析...........................................................................37
(3) BBTV其他type II分離��之Y、S1、S2及S3 library與
內含基因體之分析.......................................................37
3. BBTV type III..........................................................................37
(1) BBTV type III之Y 、S1、S2及S3 library與內含基因
體之分析.......................................................................37
4. BBTV type IV..........................................................................38
(1) BBTV type IV之Y library與內含基因體之分析......38
(2) BBTV type IV之S1 library與內含基因體之分析.....38
(3) BBTV type IV之S2 library與內含基因體之分析.....38
(4) BBTV type IV之S3 library與內含基因體之分析.....38
( ) BBTV各系統分離��additional Rep基因體之序列分析..................38
1. BBTV type I-1.........................................................................38
(1) BBTV type I-1之I-1-Ya與I-1-Yb序列分析................39
(2) BBTV type I-1之I-1-S1a與I-1-S1b序列分析.............39
2. BBTV type II-1........................................................................39
(1) BBTV type II-1之II-1-Y序列分析..............................39
3. BBTV type III之Y, S1, S2, S3序列分析.................................40
4. BBTV type IV..........................................................................40
(1) BBTV type IV之IV-Y序列分析.................................40
(2) BBTV type IV之IV-S2a與IV-S2b序列分析..............40
( ) BBTV各分離��之additional Rep序列比較與親緣演化分析..........40
1. Y、S1、S2及S3引子序列於BBTV各分離��之additional
Rep基因體之相對位置............................................................41
2. BBTV各分離��之additional Rep基因體之全長核 酸序列
比較與親緣演化分析...............................................................41
3. BBTV各分離��之additional Rep基因體轉譯之胺基酸序列
比較與親緣演化分析...............................................................42
4. BBTV各分離��additional Rep之stem-loop序列比對與結構
分析...........................................................................................42
5. BBTV各分離��additional Rep分群之病徵..........................43
、討論......................................................................................................................44
、參考文獻..............................................................................................................53
、圖表......................................................................................................................60
Tab. 1. Primer used for polymerase chain reaction..................................60
Tab. 2. Nucleotide sequences of BBTV DNA components used in
analysis................................................................................................61
Tab. 3. Nucleotide sequences of BBTV additional Rep-encoding
components used in analysis............................................................62
Tab. 4. DNA components associated with different isolates from BBTV
type I-1, II-1, III and IV......................................................................63
Tab. 5. 5’-UTR and 3’-UTR characteristics of all DNA components
among BBTV isolates, type I-1, II-1, III and IV..............................64
Tab. 6. Pairwise sequence comparison of complete nucleotide
sequences of BBTV DNA1 among isolates.....................................65
Tab. 7. Pairwise amino acid sequence comparisons encoded by major
open reading frame of BBTV DNA1 among isolates....................66
Tab. 8. Pairwise sequence comparison of complete nucleotide
sequences of BBTV DNA2 among isolates....................................67
Tab. 9. Pairwise amino acid sequence comparisons encoded by major
open reading frame of BBTV DNA2 among isolates...................68
Tab. 10. Pairwise sequence comparison of complete nucleotide
sequences of BBTV DNA3 among isolates..................................69
Tab. 11. Pairwise amino acid sequence comparisons encoded by major
open reading frame of BBTV DNA3 among isolates..................70
Tab. 12. Pairwise sequence comparison of complete nucleotide
sequences of BBTV DNA4 among isolates..................................71
Tab. 13. Pairwise amino acid sequence comparisons encoded by major
open reading frame of BBTV DNA4 among isolates..................71
Tab. 14. Pairwise sequence comparison of complete nucleotide
sequences of BBTV DNA5 among isolates..................................72
Tab. 15. Pairwise amino acid sequence comparisons encoded by major
open reading frame of BBTV DNA5 among isolates..................73
Tab. 16. Pairwise sequence comparison of complete nucleotide
sequences of BBTV DNA6 among isolates..................................74
Tab. 17. Pairwise amino acid sequence comparisons encoded by major
open reading frame of BBTV DNA6 among isolates..................74
Tab. 18. Additional Rep-encoding components associated BBTV
isolates, type I-1, II-1, III and IV.....................................................75
Tab. 19. 5’-UTR and 3’-UTR characteristics of all additional Rep-
encoding components among BBTV isolates, type I-1, II-1 and
IV........................................................................................................76
Tab. 20. Summary of PCR results using C1, S, SR, Y, S1, S2 and S3
primer pairs with different BBTV type I isolates as templates..77
Tab. 21. Summary of PCR results using C1, S, SR, Y, S1, S2 and S3
primer pairs with different BBTV type II and IV isolates as
templates...........................................................................................77
Tab. 22. Nucleotide sequences identity of BBTV additional Rep-
encoding components from I-1, II-1, Taiwan (Tw) and Vietnam
(Vt).....................................................................................................78
Tab. 23. Amino acid sequences identity of BBTV additional Rep-
encoding components from I-1, II-1, Taiwan (Tw), and Vietnam
(Vt).....................................................................................................79
Tab. 24. Nucleotide sequences identity of BBTV additional Rep-
encoding components from IV, Taiwan (Tw) and Vietnam (Vt)..
............................................................................................................80
Tab. 25. Amino acid sequences similarity of BBTV additional
Rep-encoding components from IV, Taiwan (Tw) and Vietnam
(Vt).....................................................................................................80
Tab. 26. Nucleotide sequences similarity of BBTV additional Rep-
encoding components from I-1, II-1, and IV................................81
Tab. 27. Amino acid sequences identity of BBTV additional Rep-
encoding components from I-1, II-1, and IV................................81
Tab. 28. Stem and loop sequences of BBTV additional Rep-encoding
components......................................................................................82
Tab. 29. Symptoms expression of different BBTV type I isolates in
Cavendish banana plants................................................................83
Tab. 30. Symptoms expression of different BBTV type II and IV isolates
in Cavendish banana plants...........................................................83
Fig. 1. Confirm the presence of III-L1 component by PCR reaction
using primer pairs BTV-5,6..............................................................84
Fig. 2. The alignment of III-L2 and My- Hung..........................................85
Fig. 3. Schematic representation of BBTV IVC46 component and DNA1
...............................................................................................................86
Fig. 4. Predicted diagrams of transmembrane domain of BBTV DNA
components.........................................................................................87
Fig. 5. Phylogenetic analysis of full length DNA sequence of all BBTV
genomic DNA components derived from BBTV isolates, type I,-1
II-1, III and IV......................................................................................88
Fig. 6. Alignment of DNA sequences in stem-loop region of all BBTV
genomic DNA components derived from of BBTV isolates, type
I-1, II-1, III and IV...............................................................................89
Fig. 7. Phylogenetic tree constructed from alignment of stem-loop DNA
sequences of each DNA component of BBTV type I, II, III and
IV.........................................................................................................90
Fig. 8. Alignment of nucleotides of major common region of BBTV type
I-1..........................................................................................................91
Fig. 9. Alignment of nucleotides of major common region of BBTV type
II-1.........................................................................................................91
Fig. 10. Alignment of nucleotides of major common region of BBTV
type III................................................................................................92
Fig. 11. Alignment of nucleotides of major common region of BBTV
type IV................................................................................................92
Fig. 12. Unrooted neighbor-joining trees of BBTV DNA1 based on its
sequences...........................................................................................93
Fig. 13. Unrooted neighbor-joining tree of BBTV DNA2 based on its
sequences...........................................................................................94
Fig. 14. Unrooted neighbor-joining tree of BBTV DNA3 based on its
sequences...........................................................................................95
Fig. 15. Unrooted neighbor-joining tree of BBTV DNA4 based on its
sequences...........................................................................................96
Fig. 16. Unrooted neighbor-joining tree of BBTV DNA5 based on its
sequences...........................................................................................97
Fig. 17. Unrooted neighbor-joining tree of BBTV DNA6 based on its
sequences...........................................................................................98
Fig. 18. Secondary structure of stem-loop sequence of type I-1.............99
Fig. 19. Secondary structure of stem-loop sequence of type II-1..........100
Fig. 20. Secondary structure of stem-loop sequences of type III..........101
Fig. 21. Secondary structure of stem-loop sequence of type IV............102
Fig. 22. The location of four primer pairs, SR, Y, S1, S2 on the
nucleotide sequences of additional Rep-encoding components
identified from BBTV isolates.......................................................104
Fig. 23. Alignment of nucleotide sequences of BBTV additional Rep-
encoding components...................................................................107
Fig. 24. Phylogenetic tree constructed from nucleotide sequence
alignment of total BBTV additional Rep-encoding components..
..........................................................................................................108
Fig. 25. Alignment of amino acid sequences of BBTV additional Rep-
encoding components....................................................................109
Fig. 26. Phylogenetic tree constructed from amino acid sequences
alignment of total BBTV additional Rep-encoding components..
..........................................................................................................110
Fig. 27. Secondary structure of hairpin sequences of additional Rep-
encoding components.....................................................................112
Fig. 28. Symptoms of different BBTV additional Rep groups...............114
附錄A. Agarose gel electrophoresis of different RFLP group...........................116
附錄B. Genome sequences of six integral DNA components of BBTV type I-1,
II-1, III and IV...............................................................................................120
附錄C. Genome sequences of additional Rep-encoding components of BBTV
type I-1,II-1 and IV......................................................................................161
附錄D. Genome sequences of DNA components of other BBTV type isolates....
.......................................................................................................................169

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