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標題: 甘藷長絲狀病毒鞘蛋白基因核酸序列、血清學性狀 與檢測工具研發
Coat Protein Sequence Analysis, Serological Properties and Development of Detection Tools for Two Filamentous Viruses Isolated from Sweet Potato
作者: 王麗媛
Wang, Li-Yuan
關鍵字: Sweet Potato Virus;甘藷長絲狀病毒;Coat Protein Sequence;Serological;鞘蛋白基因核酸序列;血清學性狀
出版社: 植物病理學系所
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自田間呈現黃斑、斑駁或嵌紋病徵的甘藷病株,以機械接種到指示植物奎藜 (Chenopodium quinoa),經單斑分離後可得3種大小、型態不同的病斑。因甘藷富含澱粉及酚化物等抑制物質,又無適當繁殖寄主,故分離純化不易。因此,本研究從核酸層次著手,進行解序及分析,以了解其分子特性。經與登錄於GenBank之potyvirus基因比對,得知其分別為甘藷潛伏病毒(Sweet potato latent virus, SPLV)及甘藷羽狀斑駁病毒(Sweet potato feathery mottle virus, SPFMV)。另外由鞘蛋白基因設計專一性引子對後,利用細菌載體大量表現蛋白,快速製備抗血清,供檢測用。為探討兩種病毒在田間分布情形及其複合感染對植株生長影響,需將病毒回接甘藷植株,但利用磨擦接種不易成功。本研究嘗試將甘藷株根部浸潤於病毒粗汁液,可令病毒感染成功獲得單獨感染之病株。並比較單獨感染與複合感染病株於植株中之分布,以了解於田間分布及評估與其對產量影響。
自呈現黃斑病徵甘藷病株機械磨擦接種到奎藜7天後出現壞疽病斑。由呈現壞疽病徵奎藜以簡併式引子Pot I及Oligo(dT),利用RT-PCR可增幅出2.0 kb核酸片段,經選殖及解序得全長含1930個核苷酸,與己發表之potyvirus作基因比對,得知為甘藷潛伏病毒 (SPLV),即由5''端起為831nt 細胞核內含體b (nuclear inclusion b, NIb),879 nt之全長度鞘蛋白 (coat protein, CP)基因及197 nt之3''端非轉譯區 (3'' non-coding region, 3''-NCR)及poly A尾端。與己登錄於GenBank之甘藷潛伏病毒系統 (strains)比對,結果與SPLV-TW之CP及3''-NCR之核苷酸序列相同度分別為96.5%及100%。於鞘蛋白基因兩端設計專一性引子,Lcp 1及Lcp 2兩個專一性引子,利用RT-PCR增幅出1057 nt核酸片段,經解序確定後,將其專殖到細菌表現載體pET-32a(+)中,並使其於E. Coli寄主內大量表現融合性甘藷潛伏病毒鞘蛋白。以親合性色層分析法回收純化,可得約38 kDa的融合蛋白,經免疫注射製成多元抗體。以瓊脂雙向擴散反應法測定血清力價為1/8。以間接酵素聯結抗體免疫吸附法檢測融合蛋白、壞疽病徵奎藜及斑駁或黃斑之甘藷病株,抗血清稀釋48,000倍仍可測得。以稀釋6,000倍抗血清配合西方轉漬法檢測不同病徵之田間甘藷病株,可測得33 kDa反應條帶,顯示製備之多元抗體可檢測田間罹病植物。
自呈現斑駁病徵甘藷機械磨擦接種到奎藜10-14天可出現兩種不同大小之灰白色單斑。以斑駁藷葉抽取總量核糖核酸,以簡併式引子Pot I、Pot II利用RT-PCR方法可增幅出1.3 kb及1.2 kb兩個核酸片段,經選殖及解序後與己發表之potyvirus作基因比對,得知為甘藷羽狀斑駁病毒,兩個核酸片段分別由3''端設計專一性引子將全長度鞘蛋白及3''非轉譯解序完成。解得SPFMV-CY1全長含1249個核苷酸,即比對兩條核酸序列鞘蛋白基因之相同度 (identity)為80.6%,相似度為86.3%顯示其為不同的系統 (strain),分別命名為SPFMV-CY1,SPFMV-CY2。與potyvirus屬中18個病毒比對鞘蛋白胺基酸序列,分別有50-76%相似度。與已知56個系統比對鞘蛋白核苷酸序列,SPFMV-CY1與strain C相同度最高,SPFMV-CY2則與strain O相同度最高。由SPFMV-CY1鞘蛋白基因兩端設計專一性引子FM86、FM1006,利用RT-PCR增幅出939 nt,經解序確定後,將其專殖到細菌表現載體PET-32a(+)中,並使其於E. Coli寄主內大量表現融合性甘藷潛伏病毒鞘蛋白。以親合性色層分析法回收純化,可得約35 kDa的融合蛋白,經免疫注射製成多元抗體。以間接酵素聯結抗體免疫吸附法檢測融合蛋白、灰白斑病徵奎藜及不同病徵甘藷病株,抗血清稀釋48,000倍仍可測得。以稀釋6,000倍抗血清配合西方轉漬法檢測不同病徵之田間甘藷病株,可測得35 kDa反應條帶,顯示所製備之多元抗體可檢測田間罹病植物。
於西方轉漬檢測過程中,發現SPFMV及SPLV兩個病毒之多元抗血清易呈現交互反應 (cross reaction),乃以SPFMV-CY1之融合蛋白進一步製備單元抗體。

Three kinds of single lesions with different size and morphology shows in indicator plant Chenopodium quinoa after mechanical inoculation and single lesions transfer from sweet potato with symptoms of yellow spots, vein mottling or mosaic. It is difficult to isolate and purify virus particles from sweet potato because the content of starch and phenolic compounds and the absent of the propagation host. In this study, using the RNA extract from sweet potato cloning and sequencing to search the molecular characteristics of the virus. Comparing the sequences with potyviruses genome in GeneBank evidence that the sequences are Sweet potato latent virus (SPLV) and Sweet potato feathery mottle virus (SPFMV). For antiserum preparation, the complete reading frame of the coat protein (CP) gene of SPLV and SPFMV were amplified from the total RNA extracted from virus-infected leaves of C. quinoa by RT-PCR with the cp-gene specific primers. The amplified DNA fragment was cloned, sequenced, and subcloned into the bacterial expression vector pET-32a(+) vector. For studying the influence of mix infection and distribution of viruses in field, back inoculation is necessary, but to infect sweet potato by mechanical inoculation is difficult. Therefore, the method of root dipping of sweet potato in the crude extracts of virus infected C. quinoa. Detection of the root dipping plants by western blotting indicated that these filamentous viruses can infect sweet potato through roots and the mix infection virus can be separated effectively.
The C. quinoa shows necrotic local lesions 7 dpi isolated from sweet potato with yellow spots symptoms were used as source for total RNA extraction. A 2.0-kb product was amplified from the total RNA extracted from virus-infected leaves of C. quinoa by RT-PCR with oligo(dT) and pot2 primers. The cDNA fragment reflected 1931 nucleotides (nts) corresponding to the 3''-terminal region of potyviruses was obtained. The deduced amino acid sequence contained 578 residues encoding part of the 3'-terminal region of NIb gene (285 residues) and the complete sequence of coat protein (CP) gene (293 residues). A 197 nts of non-coding region (NCR) was found located at the 3'-terminal region of the DNA. A genetic code for aphid transmissibility of potyviruses, DAG triplet, was found at the 7-9 residues from the N-terminus of CP gene. Compared to the known sequences of strains of SPLV, the percentage of nucleotide identities of the CP gene and the NCR with SPLV-T were 96.5% and 100%, respectively. Using directional cloning, a 55 kDa fusion protein containing a complete CP sequence of SPLV and a partial sequence encoded by the expression vector plasmid (pET-32a, Novagen) was expressed and purified from cell cultures of Escherichia coli. The antiserum prepared against this fusion protein showed high sensitivity in the serological detection of infected tissue of sweet potato. According to the coat protein gene of SPLV, the specific promers Lcp1, Lcp2 were designed for amplification of coat protein by bacterial expression system. The titer of prepared SPLV antiserum determined by double diffusion test is 1/8.
Two different size of chlorotic spots were shown in C. quinoa 10-14dpi. after mechanical inoculation from mottling disease symptom of sweet potato. The total RNA was extracted from mottling sweet potato, a 1.3 kb and a 1.2 kb DNA fragments were amplified by using PotI and PotII primers and RT-PCR. After cloning and sequencing and comparing to the known potyvirus in the GenBank. Specific primer was designed from the 3''-terminal of the two DNA fragments, the whole length CP and the 3''-NCR were sequenced. Total length including 1249 nts in SPFMV-CY1, the identity and the similarity were 80.6% and 86.3% comparing with CP gene of the two DNA sequences that shown they were in different strain, named SPFMV-CY1 and SPFMV-CY2, respectively. The similarity was 50-76% comparing to the 18 potyviruses with their CP amino acid sequences. The identity was highest between SPFMV-CY1 and strain C by comparing to the 56 known systems with their CP nt sequences and the similarity was highest between SPFMV-CY2 and strain O. There were 939 nts amplified by devising special primers, FM 86 and FM 1006, from both terminals of the CP gene of SPFMV-CY1 and through RT-PCR. In the proceeding of western blot, the cross reaction was found between the multi-antiserum of the two viruses of SPFMV and SPLV, and the monoclonal antibody was produced by using the fusion protein of SPFMV-CY1.
其他識別: U0005-3008200710175200
Appears in Collections:植物病理學系

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