蘭花抗病毒專一性載體構築與暫時性表達分析

Construction of Vectors Specific for Virus Resistance and Transient Expression Analysis in Orchids

葉素瑛(Su-Ying Yeh)

指導教授 ： 黃鵬林

共同指導教授 ： 杜宜殷(Dr. Yi-Yin Do)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10325

摘要

為了構築對蘭科植物(Orchidaceae)抗病毒程度較高及專一性載體，本研究應用RNA干擾技術(RNA interference, RNAi)進行表達載體構築。首先利用聚合酶連鎖反應，合成文心蘭乙烯受體基因之第三個隱子序列約150個鹼基對 (base pairs) 作為間隔區域，再以2X CaMV 35S啟動子引導，構築為RNA干擾技術用載體卡匣 (cassette)，再將喜姆比蘭嵌紋病毒及齒舌蘭輪斑病毒之外鞘蛋白基因，分別以順、反向及反、順向構築於此RNA干擾技術用載體卡匣，以獲得全長外鞘蛋白基因RNA干擾構築。另外，針對不同地區與不同蘭屬，比對CyMV與ORSV外鞘蛋白胺基酸之同源區域，選取四區25 bp構築可產生雙股small interference RNA (siRNA)之質體。
蝴蝶蘭花瓣原生質體暫時性表達分析，以表達喜姆比蘭嵌紋病毒外鞘蛋白之報導質體，與全長反、順向外鞘蛋白基因RNAi進行共轉殖，經間接式酵聯抗體法(indirect enzyme linked immunosorbent assay)測試兩者間不同分子數比例之默化能力，顯示二質體分子數比例為1:1時就可達到95%默化效果。不同表達時間進行默化測試，顯示在48小時即可達到94%默化效果。共轉殖報導質體與RNAi，或siRNA作用質體測試默化效果，表達48小時後分析，結果顯示全長RNAi構築可100%抑制喜姆比蘭嵌紋病毒外鞘蛋白之生成；而siRNA作用質體的默化效果，同源區第三區可抑制54%外鞘蛋白生成量，其他區抑制效果較差。進一步以免疫轉漬分析檢測喜姆比蘭嵌紋病毒外鞘蛋白累積量，轉殖報導質體之蝴蝶蘭原生質體蛋白經以CyMV多株抗體進行反應，可偵測出27 kDa之CyMV外鞘蛋白，若將報導質體與RNAi或siRNA作用質體進行共轉殖，則全長外鞘蛋白基因之RNAi構築可完全默化基因表現，27 kDa CyMV外鞘蛋白無法偵測到，siRNA質體以第三個同源區默化後，偵測到的CyMV外鞘蛋白量較其他區少。

關鍵字

喜姆比蘭嵌紋病毒 ； 齒舌蘭輪斑病毒 ； 小片段干擾RNA ； 基因默化 ； RNA干擾

並列摘要

The application of RNA interference (RNAi) to disrupt gene expression has become a powerful tool in plants. To construct specific RNAi vectors for virus resistance in orchids, the third intron of Oncidium ethylene receptor gene Og-ERS1 was synthesized using polymerase chain reaction (PCR). The resulting expression vector containing 2X Cauliflower mosaic virus (CaMV) 35S promoter driving RNAi cassette, was used for double-stranded RNA expression and comprised the full-length coding region sequences of Cymbidium mosaic virus (CyMV) or Odontoglossum ringspot virus (ORSV) coat protein gene in the sense and antisense configuration on either side of the intron fragment. Furthermore, four highly homologus regions of amino acid sequences of CyMV and ORSV coat protein isolated from various genera of orchids were used to construct double-stranded 25 bp small interference RNA (siRNA) expression plasmid.
Reporter plasmid encoding CyMV coat protein was co-transformed with antisense-sense RNAi effector plasmid via transient expression analysis in protoplasts from Phalaenopsis petals. The 1:1 molecule ratios between CyMV coat protein reporter plasmid and RNAi effector plasmid had 95% silencing effect using indirect enzyme linked immunosorbent assay for analysis. The proper time point to analyze the silencing effect of CyMV coat protein RNAi effector plasmid was 48 hr after co-transformation for 94% silencing. The silencing effect of full length CyMV coat protein RNAi construct was 100%. The production of coat protein was reduced by 54% for siRNA expression construct containing the third homologous region detected 48 hr after co-transformation. Immunoblot assays showed that CyMV coat protein expressed in transformed protoplasts from Phalaenopsis petals with the expected molecular mass of 27 kDa. No detectable CyMV coat protein in protoplasts co-transformed with reporter plasmid and RNAi effector plasmid was found. Decreased levels of coat protein were accumulated in protoplasts co-transformed with reporter plasmid and the siRNA expression construct containing third homologous region

並列關鍵字

Odontoglossum ringspot virus ； Cymbidium mosaic virus ； small interference RNA ； RNA interference ； gene silencing

參考文獻

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