Development of Identification Chip for Important Solanaceous Plant Viruses

茄科作物為重要的蔬菜和水果，生長期間常遭受病毒危害，且田間的植株可能會被一種以上的病毒複合感染。依據國外資料記載，可感染茄科作物的病毒種類超過20屬80種以上，而國內也有40種以上的病毒可以感染茄科作物，其中又以cucumoviruses、potyviruses和tobamoviruses最為嚴重，對作物造成重大損失。在面對這些可能遭受各種病毒感染的作物，常使用的檢測方法有ELISA、RT-PCR等，但為達成一次實驗可同時診斷多種病害的目標，我們將生物晶片應用於茄科病毒之鑑定，以cucumoviruses、potyviruses和tobamoviruses作為診斷鑑定晶片的檢測對象。我們的研究策略需要設計簡併性引子對(degenerate primer)，因此先從GenBank收集相關病毒的胺基酸序列，再利用ClustalX程式進行胺基酸序列多序列並列比對，根據比對結果利用CODEHOP軟體找出具有保守性的區域，再依照該軟體的策略設計簡併性引子。之後以簡併性引子對所圍成的區域，找出病毒種間差異性較大的區段，設計專一性寡核苷酸探針(oligonucleotide probe)。請廠商合成50 mer寡核苷酸探針之後點漬在載體上，載體可為尼龍膜或是經過處理的玻片，再以UV光照射固定之。利用Potyvirus、Tobamovirus和Cucumovirus三屬病毒的簡併性引子對，配合actin引子對，以植物樣品全RNA進行multiplex RT-PCR。所增幅並已DIG標定的核酸產物，作為標的物，然後對製備好的晶片進行雜合反應和結果判讀。目前已針對這三屬病毒設計出十四種探針，且由雜合反應結果可知，針對所測試的植物樣品，不論是單獨感染或是複合感染病毒的樣品，皆可被茄科病毒診斷鑑定晶片正確的鑑定出來。此診斷鑑定晶片具有擴充能力，未來可再增加病毒探針數目及種類，以供不同研究目的使用。Solanaceous plants include many important vegetables and fruits such as tomato, pepper, potato, etc. There are many kinds of plant viruses which can infect solanaceous plants and mixed infection happens frequently in the field. According to the literatures, more than 20 genera 80 species viruses infect solanaceous plant. In Taiwan, there are over 40 viruses including tobamoviruses, potyviruses and cucumoviruses reported to infect solanaceous plants and cause heavy losses. ELISA and RT-PCR are commonly used methods for plant virus detection. Nevertheless, it will be preferable to have a specific and sensitive method which can detect many viruses simultaneously. Therefore, a solanaceous virus chip based on the principle of DNA microarray was attempted to develop. At first we download viral amino acid sequences from GenBank. After using ClustalX software to align amino acid sequences, we used CODEHOP software to separately design degenerate primers of potyviruses, tobamoviruses and cucumoviruses from aligned amino acid sequences. After that, species-specific oligonucleotide probes were designed from the regions between each degenerate primer pair. DIG-labeled or Cy3-labeled targets were prepared from total RNA of infected tissues by means of multiplex RT-PCR with all sets of degenerate primers. Oligonucleotide probes were immobilized onto nylon membrane or slide chip, hybridized with the targets and then the hybridization signals were detected. The results indicated that solanaceous virus chip could correctly identify potyviruses, tobamoviruses and cucumoviruses in single and mixed infection plants. Up to the present, we have designed 14 virus probes for three genera of plant viruses. This virus chip is expandable to include new probes which are required for particular purpose.目錄………………………………………i 誌謝………………………………………ii 中文摘要…………………………………1 英文摘要…………………………………2 前言………………………………………3 前人研究…………………………………3 生物晶片…………………………………5 病毒介紹…………………………………6 標定方式…………………………………9 材料方法…………………………………11 實驗植物…………………………………11 病毒材料…………………………………11 收集序列資料並設計簡併性引子對……12 種專一性寡核苷酸探針的設計與製備…13 反轉錄反應………………………………14 聚合酶連鎖反應…………………………14 多對引子反轉錄聚合酶連鎖反應………15 標的物之製備……………………………15 逆墨點雜合反應…………………………16 結果………………………………………20 討論………………………………………26 參考文獻…………………………………30 圖…………………………………………3