臺灣博碩士論文加值系統

朱槿（Hibiscus rosa-sinensis）為錦葵科（Malvaceae）木槿屬（Hibiscus）之多年生常綠灌木，原產於熱帶亞洲地區。生長茂密，花大而艷麗，適合作為籬笆及盆栽種植。於高師大校園及農委會種苗場取得之朱槿植株，發.現有明顯壞疽及嵌紋病徵，進行病原鑑定，證實受到Hibiscus latent Fort Pierce virus（HLFPV）及Hibiscus latent Singapore virus（HLSV）兩種病毒的複合感染。經三次單斑分離獲得純系病毒分離株H1及H2´。H1為HLSV分離株；H2´為HLFPV分離株，並命名為HLFPV臺灣分離株（HLFPV-TW）。本實驗的目的在利用反轉錄聚合酶連鎖反應(RT-PCR)進行HLFPV-TW基因序列的譯讀，並與National Center for Biotechnology Information （NCBI）網站上GenBank之HLFPV-U（美國，Accession number：CP_ AY250831, MP_ AY596456, 128/186 kDa_ FJ196834）、HLFPV-Hybrid29（泰國，Accession number：CP_AY560557）、HLFPV-rosa15（泰國，Accession number：CP_ AY560554）、HLSV-S1（新加坡，Accession number：FJ196834, AY596456, AY250831）、HLSV-S2（新加坡，Accession number：NC_008310）進行核苷酸和胺基酸序列比對與分析。經譯讀後共解得6,224個核苷酸（不包括5´端非轉譯區及3´端非轉譯區），存在四個開放轉譯架構（open reading frame, ORFs），分別為RdRp（RNA-dependent RNA polymerase）/ replicase protein、RdRp/readthrough protein、MP（movement protein）和CP（coat protein），從5'到3'端方向依序有3,417個核苷酸，蛋白為128.5 kDa（1,138 aa）、4,941個核苷酸，186.6 kDa （1,646 aa）、855個核苷酸，31.1 kDa（284 aa）和477個核苷酸，17.6 kDa（158 aa），其中replicase protein和 readthrough protein這兩個蛋白起始位置相同，readthrough protein基因和MP基因以及MP基因和CP基因都有重疊。綜合來說， HLFPV-TW與HLFPV-U、HLFPV-Hybrid29、HLFPV-rosa15間的多重核苷酸序列及胺基酸序列比對皆展現高度同源性，種內遺傳歧異度相當低。而HLFPV-TW與HLSV-S1、HLSV-S2差異性較大，為同一病毒屬之不同種。

Chinese hibiscus (Hibiscus rosa-sinensis), which are native to Tropical Asia, a species of flowering plant in the family Malvaceae, are the perennial plants of evergreen bushes with dense growth and attractive flowers is suitable for hedges or potted plants. The Chinese hibiscus with significant necrotic and mosaic symptoms were collected from Campus of NKNU and Seed Improvement and Propagation Station. They were complex infection by Hibiscus latent Fort Pierce virus (HLFPV) and Hibiscus latent Singapore virus (HLSV) through identification of virus. The virus isolated (H1 and H2´) were obtained via three successive single lesion isolations on Chenopodium quinoa. H1 isolated was infected with HLSV; H2´ isolated was infected with HLFPV, and to give a name to HLFPV-TW. This study aimed to sequence nearly full length of the viral genome of Hibiscus latent Fort Pierce virus Taiwan isolate (HLFPV-TW), and compare the nucleotide and amino acid sequences with HLFPV-U (USA, accession number：CP_ AY250831, MP_ AY596456, 128/186 kDa_ FJ196834) , HLFPV-Hybrid29 (Thailand, accession number：CP_AY560557) , HLFPV-rosa15 (Thailand, Accession number：CP_ AY560554), HLSV-S1 (Singapore, accession number：FJ196834, AY596456, AY250831) and HLSV-S2 (Singapore, Accession number：NC_008310) in the GenBank of NCBI have been carried out. As the result of sequencing, nearly full length of the viral genome were 6,224 nucleotides (exclusive of 3´ NTR and 5´ NTR ). From 5' to 3' end, four open reading frames (ORFs) were recognized as RdRp/replicase protein, RdRp/readthrough protein, MP and CP genes. RdRp/replicase gene was 3,417 bp which encoded a protein of 128.5 kDa (1,138 aa), RdRp/readthrough gene was 4,941 bp which encoded a protein of 186.6 kDa (1,646 aa), MP gene was 855 bp which encoded a protein 31.1 kDa (284 aa) and CP gene was 477 bp which encoded a protein of 17.6 kDa (158 aa), respectively. Two proteins of RdRp were initiated at the same site. There were several overlaps between RdRp/readthrough genes and MP genes, and between MP genes and CP genes. In conclusion, the comparison of multiple alignments of nucleotide and amino acid sequences of HLFPV-TW with HLFPV-U, HLFPV-Hybrid29, and HLFPV-rosa15, displayed high identities in intraspecies. In the phylogenetic analysis, they had a closer relationship. However, the nucleotide and amino acid sequences of HLFPV-TW with HLSV-S1 and HLSV-S2 were quietly different from others. They belong to the same genus but not of the same species.

目錄

中文摘要 I
英文摘要 III
壹、前言 1
一、朱槿簡介 1
二、可感染朱槿之病毒種類 3
三、Hibiscus latent Fort Pierce virus（HLFPV）病毒簡介 4
四、Tobamovirus屬病毒特性 6
五、研究目的 12
貳、材料與方法 14
一、病毒的來源與初步鑑定 14
(一) 病毒的來源 14
(二) 植物 total RNA之萃取 14
(三) 反轉錄聚合酶連鎖反應 15
(四) 病毒的分離 16
二、HLFPV臺灣分離株（HLFPV-TW）核苷酸序列之增幅與選殖 17
(一) 反轉錄聚合酶連鎖反應（Reverse transcription - polymerase chain reaction, RT-PCR） 17
(二) 回收PCR產物 19
(三) 接合作用（Ligation） 19
(四) 轉形作用（Transformation） 19
(五) 篩選正確的質體DNA 20
三、HLFPV-TW核苷酸序列之譯讀與分析 21
(一) 核苷酸序列之譯讀 21
(二) 核苷酸序列之分析與比對 21
四、HLFPV-TW與國外已發表之同種病毒及Hibiscus latent Singapore virus（HLSV）進行核苷酸及胺基酸序列之比較 22
參、結果 23
一、HLFPV-TW病毒的來源與分離 23
(一) 病毒以反轉錄聚合酶連鎖反應之初步鑑定 23
(二) 病毒的的分離 23
二、HLFPV-TW核苷酸序列之定序與分析 25
(一) 反轉錄酶連鎖反應之電泳膠體分析 25
(二) 用PCR篩選出正確的質體DNA之電泳膠體分析 25
(三) HLFPV-TW核苷酸序列之譯讀與分析 26
三、HLFPV-TW與國外同種病毒及HLSV之核苷酸及胺基酸序列比較 27
肆、討論 29
伍、圖表 37
陸、參考文獻 64
柒、附錄 73

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