臺灣博碩士論文加值系統

摘要
洋桔梗壞疽病毒(Lisianthus necrosis virus, LNV) 最早於1983年由日本Iwaki 等人所發現，但因LNV的核酸序列及基因體的組成尚未被發表，故LNV依據血清學及其物理特性，暫被分類在Tombusviridae科Necrovirus屬。晚近在台灣中部彰化地區栽培之洋桔梗(Eustoma rusellianum (Don.) Grieseb)以及進口康乃馨(Dianthus caryophyllus L.)雜交品系”小可愛(Kooij Echo kgr. Holland)”上亦發現受LNV感染。LNV通常感染生育中、後期之植株，造成於上位葉出現許多淡黃色斑點，鄰近斑點隨病勢進展融合成斑塊，呈黃色之組織逐漸轉化為壞疽，終致全葉枯萎。本研究之目的在於選殖、闡明LNV基因體序列及組成，並確認或重新定位LNV的分類地位。本研究將台中改良場陳慶忠博士自永靖鄉洋桔梗罹病株取得並經過三次單斑分離之LNV洋桔梗分離株(LNV-L) 繁殖於其系統性寄主菸草(Nicotiana benthamiana Domin.)上，以西方轉漬法利用LNV抗血清偵測經純化之病毒，確定其鞘蛋白大小約為41 kDa；於電子顯微鏡下觀察並測量其病毒顆粒為直徑32 nm的球形病毒。另外為了獲得LNV基因體序列，以經抽取、純化的總量RNA (total RNA)及雙股核糖核酸(dsRNA)做為反轉錄聚合反應(reverse transcription-polymerase chain reaction, RT-PCR)的模板；利用由Necrovirus之核酸序列高度保留區(highly conserve region)設計的兩組簡併式引子對(degenerated primers)增幅出部份LNV cDNA片段，此片段經選殖、定序及序列分析之結果與新發現暫分類於Tombusvirus屬的梨潛隱病毒(Pear latent virus, PLV)有高度相似性；並利用所得之序列與PLV及其他Tombusvirus屬病毒之序列比較，設計簡併式或專一性引子以獲得洋桔梗壞疽病毒的全基因體序列。LNV全基因體序列主要包含在三個利用RT-PCR增幅所得之選殖株中。基因體大小全長為4764個核苷酸(nucleotide, nt)，對應轉譯出五個蛋白。第一個轉譯架構（open reading frame, ORF 1）長約2457 nt，對應產生一與病毒複製相關之RNA複製酶（RNA-dependent RNA polymerase），分子量大小約為92 kDa。ORF 2長約1167 nt，對應產生一分子量大小約為41 kDa的鞘蛋白(coat protein)。ORF 3長約576 nt，對應產生分子量大小約22 kDa的蛋白，ORF 4長約519 nt，對應產生分子量大小約19 kDa的蛋白，另外在基因體的最3端則另外對應產生一3.2 kDa的鞘蛋白。此基因體的組成與Tombusvirus核酸序列相同度在73.2%至97.2%之間，尤其是和PLV的序列相同度可高達97.2%。由於核酸序列相同度以及胺基酸序列相同度皆高於95%，因此LNV與PLV可能為分類地位極相近之病毒；此外，基於LNV與Tombusvirus屬病毒及PLV核酸胺基酸序列相同度和基因體組成的相似性，我們認為LNV應被重新分類於Tombusvirus屬。

Lisianthus necrosis virus (LNV), a positive single-stranded RNA virus, was first found in Japan in 1983 and later in Taiwan in 1995. LNV causes severe necrotic spots on leaves and stems, colored stripes on petioles, and malformation of flowers in both lisianthus〔Eustoma russellianum (Don.) Griseb〕and carnation (Dianthus caryophyllus L.). LNV was delineated as a tentative species of the genus Necrovirus based on its physical, antigenic, and biological properties. In order to classify the taxonomic status of LNV, molecular characteristics including the genome organization and the sequence of coat protein gene are needed. So far there is no molecular information of LNV available yet. The objectives of this study were to clone and elucidate the genome organization and genomic sequence of LNV and to confirm or re-classify its taxonomic status. Virions purified from LNV-L-infected plants revealed the size of the particles measuring about 32 nm in diameter under electron microscope. Single coat protein with Mr. about 41 kDa was observed from the purified virions assayed by SDS-PAGE and immunoblotting. In order to obtain sequence information of LNV, two pairs of degenerate primers for Necrovirus were designed. One cDNA fragment around 700 base pairs (bp) was amplified from the total RNAs extracted from LNV-infected N. benthamiana by RT-PCR using the degenerate primer pairs Fjj2003-13/ Fjj2003-14 and Fjj2003-15/Fjj2003-16 in combination. This 700 bp RT-PCR product was cloned into the pCRII-TOPO vector, then sequenced and was found to have more than 90% nucleotide identities with those of Pear latent virus (PLV), a putative species of Tombusvirus. Three distinct cDNA fragments covering most of the LNV genome except the 5’- and 3’-termini were obtained by RT-PCR using degenerate primers designed from PLV and tombusviruses. The sequences of the 5’ and 3’ termini were obtained from RT-PCR using oligo-d (T) as primer with the polyadenylated dsRNA as template. The complete genomic sequence of LNV was then determined. The genome of LNV consists of 4764 nucleotides (nt) with five open reading frames (ORFs). ORF 1 around 2457 nt long encodes a 33.2-kDa protein and read-through of its amber termination codon gives a protein of 92 kDa. ORF 2 measuring around 1167 nt encodes a 41-kDa protein. ORF 4 is nested within the ORF 3 in a different reading frame. The molecular weight of the protein encoded by ORF 3 and ORF 4 are 21 and 19 kDa respectively. ORF 5 is located at the 3’ terminus of the genome with a molecular weight of 3.2 kDa. The genome organization of LNV was found to be similar to that of Tombusvirus, showing a nucleotide identity ranging from 73.2% to 97.2%, with the highest nucleotide identity (97.2%) to those of Pear latent virus (PLV). The phylogenetic analysis of the coat protein of LNV with other viruses in the Tombusviridae revealed that LNV is closely related to Tombusvirus rather than Necrovirus. The phylogenetic analysis of the coat protein of LNV with those of the other viruses in the Tombusvirus revealed that the LNV is closely related to the PLV. Taken together, based on nucleotide and amino acid identities of the CP gene and genome organization between LNV, Tombusvirus, and PLV, we suggest that LNV should be re-delineated from Necrovirus into Tombusvirus.

Content
Abstract in English….……………………………………………….…1
Abstract in Chinese………….………………………………………….3
Literature Review…………………………………………………….…5
Introduction………………………….……………………………..….11
Materials and Methods………..………………………………………18
Virus sources and propagation…………………….………….……. 18
Purification of virus particles……...……….………….……………18
Electron microscopy……………………………………………...…19
Western blotting…………………………………………………….19
Isolation of total RNA……………………………………………....20
Design of degenerated primers of Necrovirus and Carmovirus…….21
Reverse transcription-polymerase chain reaction (RT-PCR)……......23
DNA cloning and sequencing…………………………………….....23
Isolation of dsRNAs………………………………………………...24
Separation of dsRNAs…………………………………………...….25
Northern blot analysis…………………………………………...…..25
Cloning of the 5’ and 3’ end regions…………………………….......26
Polyadenylation of LNV dsRNA………………………………..…..27
Cloning of the 5’ and 3’ terminal regions…………..,………………28
Designing of specific primers for cloning 1871-3222 region……….29
Sequencing and assembly…………………………………….…..…29
Computer analysis of the sequences……………………………...…29
Phylogenetic analysis of the coat protein………………………...…30
Results…………………………………………………………………..32
Purification of virus particles…….....……………………………....32
Sequencing the clone obtained from the degenerate primers……...32
Isolation and separation of dsRNAs…………………………….......33
Northern blot analysis……………………………………………….34
Complete nucleotide sequence of LNV-L…………………………..34
Comparison of the nucleotide and amino acid identity between
LNV-L and other species in the Tombusvirus………………...36
Discussion………………………………………………………………38 References………………………………………………………………43
Figures and Tables……………………………………………………..52

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