臺灣博碩士論文加值系統

中文摘要
台灣新發現的彩色海芋黃化斑點病毒 (Calla lily chlorotic spot virus, CCSV) 經由S RNA 全長、L RNA 高度保留區域的解序比對，以及製備多元抗體、單元抗體偵測CCSV與其他Tospovirus屬病毒-西瓜銀班病毒 (Watermelon silver mottle virus, WSMoV) 的交互反應，已證實CCSV為蕃茄斑萎病毒屬中西瓜銀班病毒血清群 (WSMoV serogroup) 之ㄧ員。為了進一步研究CCSV在分子層次上的特性，本研究對其M 及L RNA 進行解序及分析。CCSV M RNA 之核苷酸序列，全長4,704個核苷酸，其RNA具有雙極性蛋白轉譯策略，病毒股對應產生34.6 kDa的非結構性蛋白 NSm，而其互補股則對應產生127.5 kDa的醣蛋白 GN/GC。其NSm及GN/GC蛋白，與番茄斑萎病毒 (Tomato spotted wilt virus, TSWV)、鳶尾花黃斑病毒 (Iris yellow spot virus, IYSV)、鳳仙花疽斑病毒 (Impatiens necrotic spot virus, INSV)比對，僅有29-62%的相同度；而與同屬於WSMoV serogroup的WSMoV、花生頂芽壞疽病毒 (Peanut bud necrosis virus, PBNV)、辣椒黃化病毒 (Capsicum chlorotic virus, CaCV)，以及親緣關係相近的甜瓜黃斑病毒 (Melon yellow spot virus,MYSV) 比對，則有64-81%較高的相同度。分析CCSV與其他tospoviruses GN/GC蛋白的轉譯後修飾，CCSV與WSMoV, PBNV, CaCV, MYSV之GN/GC蛋白發生裂解位置 (Signal peptidase cleavage site)、膜穿透區域 (Transmembrane region)，醣苷化位置 (N-linked, O-linked glycosylation site)以及胺基酸序列均較其他番茄斑萎病毒群 (tospoviruses) 相近，顯示CCSV屬於WSMoV 血清群 (WSMoV serogroup)。此外，TSWV，番茄黃斑病毒 (Tomato chlorotic spot virus, TCSV)，落花生輪斑病毒 (Groundnut ringspot virus, GRSV)，INSV的GN/GC蛋白皆具有與薊馬傳播相關的胺基酸保留區域 (精胺酸-甘胺酸-天冬胺酸) (Arginine-glycine-aspartic acid, RGD domain)；而CCSV, WSMoV, PBNV, CaCV, IYSV則不具有RGD domain，推測可能與傳播不同血清群病毒之薊馬種類不同有關。CCSV L RNA經解序後，全長8,911個核苷酸，其RNA具有負極性轉譯蛋白策略，病毒互補股對應產生322 kDa的複製酶 (RdRp) (L protein)。其 L蛋白 (L protein) 與TSWV，INSV比對，僅有43-45%相同度；而與WSMoV, PBNV, CaCV, MYSV比對，則有74-77%的較高相同度。分析CCSV與其他tospoviruses L蛋白的胺基酸序列，皆具有已報導過的病毒複製酶高度保留區域 A, B, C, D, E, F (RdRp conserved motifs A-F)，也發現1, 2, 3, 4, 5, 6 胺基酸序列高度保留區域 (L protein conserved regions 1-6)。從複製酶的演化關係圖可見，CCSV與WSMoV serogroup最相近；而Tospovirus屬與Bunyaviridae 科中Bunyavirus屬的關係較與Phlebovirus 屬及Hantavirus屬的關係要接近。此外，在CCSV與其他tospoviruses L RNA上的比對，五個L RNA 序列保留區域a, b, c, d, e具有21個高相似度的核苷酸，具有設計成人工微核糖核酸 (artificial miRNA) 以建構基因轉殖作物的潛力，達到作物廣泛抗tospoviruses的目的。經由CCSV與其他tospoviruses L RNA上的比對，我們設計三組簡併式性的引子 (degenerate primers)，分別是根據L基因高度保留區2以及高度保留區F所設計的引子對t2740, t3920; 及根據NSm基因高度保留區所設計的引子對tNSm410, tNSm870能夠分別增幅出不同血清群的tospoviruses: WSMoV, CaCV, CCSV, MYSV, TSWV, GRSV, INSV, IYSV, 番茄黃果輪點病毒 (Tomato yellow fruit ring virus, TYFRV)其L gene與 NSm geneg上的核苷酸片段, 大小分別為1.18 kbp與0.46 kbp。 使用tNSm410, tNSm870還能夠增幅出落花生黃化扇斑病毒(Peanut chlorotic f an-spot virus,PCFV)上較其他tospoviruses NSm片段小的DNA產物，可能是因為PCFV親緣關係較其他tospoviruses遠；因此tNSm410, tNSm870的偵測效果較t2740 t3920廣泛，效果也較優良。而根據GNGC基因高度保留區所設計的引子對WG1, WG960可以增幅出WSMoV血清群的tospoviruses: WSMoV, CaCV, CCSV, 以及IYSV血清群的tospoviruses: IYSV和TYFRV, 和MYSV其GNGC gene上的核苷酸片段，大小約為0.96 kbp；其他如TSWV血清群的tospoviruses: TSWV, GRSV, 以及INSV血清型、PCFV血清型均無法被偵測。因此，使用WG1, WG96能夠區別亞洲型與歐美型的病毒。此三組簡併式引子對可用來檢測田間罹患各種不同tospoviruses的作物，深具應用潛力。

ABSTRACT
Calla lily chlorotic spot virus (CCSV) has been classified as a distinct tospovirus belonging to Watermelon silver mottle virus (WSMoV) serogroup. To further characterize the relationships of CCSV with members of WSMoV serogroup, the nucleotide sequences of M RNA and L RNA of CCSV were determined by cloning and sequencing the cDNA fragments amplified by reverse transcription-polymerase chain reaction (RT-PCR). The complete sequence of CCSV M RNA contains 4,704 nucleotides in length. The CCSV M RNA encodes a nonstructural (NSm) protein of 309 amino acids in the viral strand, and a GN/GC protein precursor of 1,123 amino acids in the viral complementary strand. The NSm protein of CCSV shares amino acid identities of 37-39% with those of TSWV and INSV, 57-72% with those of MYSV, IYSV, WSMoV, CaCV and PBNV. In additon, the GN/GC protein precursor of CCSV shares amino acid identities 32-34% with those of TSWV and INSV, 64-73% with that of MYSV, CaCV, WSMoV, and PBNV. Our results indicate that both NSm protein and GN/GC protein of CCSV share higher amino aicd identities with those of WSMoV serogroup tospoviruses. The complete sequence of CCSV L RNA contains 8,911 nucleotides in length. The L RNA is of negative polarity, encoding L protein in the viral complementary strand with a predicted Mr of 332 kDa. L protein of CCSV shares low amino acid identities of 43-45% with those of TSWV and INSV; but it shares higher amino acid identities of 74-77% with those of MYSV, CaCV, PBNV, and WSMoV. Comparison of the deduced L protein of CCSV with that of the other members of the family Bunyaviridae indicated that its amino acid sequence include six conserved motifs A, B, C, D, E, and F of RNA-dependent RNA polymerases (RdRp). Additional conserved regions among tospoviral regions 1, 2, 3, 4, 5, and 6 were also identified. A phylogenetic cladogram of the RdRp indicated that CCSV belongs to the WSMoV serogroup of the genus Tospovirus and has a closer relationship with the viruses of genus Bunyavirus than with those of the genera Phlebovirus and Hantavirus. Multiple nucleotide alignments of L RNAs of CCSV with those WSMoV, PBNV, CaCV, MYSV, TSWV and INSV showed that there are several L RNA conserved regions at the nucleotide level among WSMoV serogroup or all tospoviruses. Five L RNA conserved regions a, b, c, d, and e containing 21 conserved nucleotides were suggested as an artificial micro RNA targeting sites for construction of transgenic crops with broad-spectrum resistance against tospoviruses. The degenerate primer pairs t2740/t3920 and tNSm410/tNSm870 were designed from the conserved regions of L RNA of WSMoV, PBNV, CaCV, CCSV, MYSV, TSWV, INSV and the conserved regions of NSm ORFs of WSMoV, PBNV, CaCV, CCSV, MYSV, TSWV, TCSV, GRSV, INSV, respectively, for amplification of the targeted regions of tospoviruses. The positions of degenerate primer t2740 was located in RdRp conserved region 2, and t3920 in conserved motif F. Tospoviruses with different serogroups or serotypes: WSMoV, CCSV, CaCV, MYSV, TSWV, GRSV, INSV, IYSV, and TYRV were detected by using the primer pairs. The amplified product obtained from PCFV-infected N. benthamiana was formed with size less than 0.46 kbp by using tNSm410/tNSm870. This may due to PCFV has no serological relationship with other tospoviruses. Our results indicated that degenerate primer pair tNSm410/tNSm870 have a better ability for broad-spectrum detection of tospoviruses from field crops. Degenerate primer pair WG1/WG960 were designed from the conserved regions of GNGC ORFs of WSMoV, PBSV, CaCV, CCSV, MYSV, and IYSV for amplification of a 0.96 kbp GNGC fragment of WSMoV, IYSV serogroup tospoviruses, or MYSV by RT-PCR. No cDNA products were obtained from TSWV, INSV, GRSV, or PCFV-infected plants of N. benthamiana. Degenerate primer pair WG1/WG960 have a potential to be used as a diagnosis tool to differentiate Asian type tospoviruses from European type tospoviruses.

目 錄

中文摘要 1

英文摘要 3

Literature Review 5

Discovery and classification of tospovirus 5

Morphology and genome organization of tospovirus 5

Tospoviruses species and taxonomy 6

Maturation of tospoviruses 8

Relationship of thrips with the tospoviruses 10

Discovery and classification of Calla lily chlorotic spot virus (CCSV) 14

Host range of CCSV 16

Introduction 17

Materials and Methods 21

Virus sources and propagation 21

Isolation of total RNA 21

Reverse transcriptation-polymerase chain reaction (RT-PCR) and cloning of the cDNA of M RNA and L RNA 21

Cloning the 5''- and 3''- terminal regions of CCSV M RNA and L RNA 24

Sequence analysis of M RNA of CCSV and L RNA 25

Post-translational modification of CCSV Glycoproteins 26

Detection of different serogroup tospoviruses by RT-PCR
using degenerate primers by RT-PCR 27

Results 29

Nucleotide sequence of CCSV M RNA and L RNA 29

Characteristics of terminal sequences and the intergenic region 30

Comparison of the NSm and GN/GC genes with those of other tospoviruses 30

Comparison of the RdRp gene with those other tospoviruses 31

Conserved L RNA regions of tospoviruses for artificial miRNA designing 33

Detection of different serogroup tospoviruses by RT-PCR using degenerate primers by RT-PCR 33

Post-translational modification of CCSV Glycoproteins 34

Discussion 35

References 42

Figures and Tables 54

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