透過您的圖書館登入
IP:54.242.96.240
  • 期刊

感染菊花之番茄種子不稔病毒之分子與血清檢測

Molecular and Serological Detection of Tomato Aspermy Virus Infecting Chrysanthemum in Taiwan

摘要


菊花為國際性重要之花卉作物,其種苗以扦插為主要繁殖方式,故必須避免系統性病原的感染。已知的系統性病原中番茄種子不稔病毒(Tomato aspermy virus,簡稱TAV)可造成感受性菊花品系花朵畸形而影響商品價值。本研究根據已知之TAV鞘蛋白基因(CP)之上下游區域設計一專一性引子對(TAV-up/TAV-dw),於反轉錄聚合酵素連鎖反應(R-PCR)下,可穩定地由彰化地區所採集之菊花樣品中增幅出一個符合預期780bp之DNA產物。此產物經選殖及核酸定序分析確定其全長為776個核苷酸,與登錄於GenBank之 TAV CP基因(D01015)有高達92%之相同度,且渠等之胺基酸序列相同度亦高達93%,此結果證實TAV已存在於台灣菊花。為獲得純化TAV鞘蛋白以製備病毒偵測用抗體,本研究採取利用細菌表現病毒鞘蛋白之策略,根據所得之TAV鞘蛋白基因序列設計分別包含Nco I及Xho I限制酵素切位之專一性引子對(TAV-uP1及TAV-dw1),將TAV CP之轉譯架構加以增幅並構築於蛋白表現載體pET-28b(+)上,其後將其轉型於Escherichia coli strain BL21(DE3)pLysS寄主內進行蛋白表現;經IPTG誘導結果得到一31kDa之表現蛋白,經西方轉漬法證實此蛋白可與商業化TAV抗體(Agdia,Inc.,Elkhart,IN,USA)反應,確定為TAV之鞘蛋白。將此蛋白大量純化後進行每隔一週連續四次之紐西蘭白兔免疫注射以製備抗血清。此抗血清可應用於酵素聯結抗體免疫吸附法(ELISA)偵測田間菊花樣品之TAV感染,與商用Agdia TAV抗體比較,對同一稀釋倍數之田間菊花樣品本抗體所測得之反應值均高於與Agdia TAV抗體之反應值,對健康菊花樣品之背景值也較低。此結果顯示以此策略所製備之抗體反應性已不亞於商業應用抗體。但本研究所製備之TAV抗體於ELISA下仍會與TAV具親綠性之胡瓜嵌紋病毒(Cucumber mosaic virus,CMV)發生交叉反應(cross reaction);反之,本研究室過去所製備之CMV抗體並不會與興TAV菊花樣品反應。此結果顯示應用血清試驗偵測TAV時需以CMV抗體同時進行雙向試驗(reciprocal tests)方能獲得正確鑑別結果。另外以前述TAV專一性引子對(TAV-up/TAV-dw)於RT-PCR反應下只能與TAV反應增幅出預期之780bp產物,而不會與CMV產生任何增幅反應。證實興TAV與CMV雖具血清類綠關係,但二者分子特性上仍有明顯差異。

並列摘要


Chrysanthemum is an ornamental crop with significant international importance. Tomato aspermy virus (TAV) is known to induce malformation of floral parts on sensitive chrysanthemum cultivars jeopardizing the quality and yield. We designed a set of primer (TAV-up/TAV-dw) according to TAV's coat protein (CP) gene sequence documented in the GenBank (accession No. D01015) and successfully amplified a 780 bp DNA product by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) from chrysanthemum specimens collected from Chang-Hua County, the major chrysanthemum production area in Taiwan. The product was cloned and sequenced. It was found to consist of 776 nucleotides (nts) corresponding to the genome organization of the 3'-terminal region of RNA 3 of cucumoviruses. The only open reading frame deducing in this sequence contains 220 amino acid residues coinciding to the size of reported TAV CP. Comparing with the known sequence of TAV CP (D01015), the percent identity of nucleotide and amino acid sequences are 92% and 93%, respectively. This result indicates the sequence amplified from chrysanthemum is originated from a strain of TAV. To our knowledge, this is the first report of the occurrence of TAV in Taiwan. In order to produce antiserum against TAV for the purpose of virus identification, we took the approach of cloning and expressing the TAV CP gene in bacteria and using the bacteria expressed CP as immunogen for antiserum preparation. By the use of directional cloning techniques, a 31 kDa fusion protein containing entire TAV CP sequence was highly expressed and purified from E. coli cell cultures. An antiserum (TAV-CP Ab) was prepared against the expressed TAV CP and shown to be useful in ELISA for the detection of TAV in chrysanthemum plants. By comparing with commercialized TAV antiserum (Agdia Inc. Elkhart, IN, USA), the reactivity in terms of ETA readings of TAV-CP Ab to the same dilution of infected chrysanthemum tissue was always higher than that of Agdia's TAV antiserum. Our results also showed TAV-CP Ab, as well as Agdia's TAV antiserum, cross reacted with Cucumber mosaic virus (CMV), another commonly found species of Cucumovirus. However, no cross reactivity to TAV was found using antiserum to CMV in a reciprocal ELISA test, showing that it is necessary to perform reciprocal ELISA tests to distinguish TAV from CMV correctly. On the other hand, our study showed TAV could be readily differentiated from CMV by RT-PCR using the aforementioned primer set (TAV-up/TAV-dw). This primer set does not amplify any product from the RNA template of CMV but consistently obtains a 780 bp product from TAV.

延伸閱讀