抗木瓜輪點病毒的基因轉殖木瓜高效能鑑別方法之研發與應用

Abstract

本研究主要以各項DNA檢測技術為基礎，建構一套完整的基因轉殖木瓜篩檢網，以防止抗木瓜輪點病毒（Papaya ringspot virus = PRSV）的基因轉殖木瓜與非基因轉殖木瓜混雜難辨的情況產生，進而提供國內消費市場與國際行銷通路上的標示依據。本論文所研發的方法涵括：一、PCR （polymerase chain reaction）檢測法：主要針對35S promoter、PRSV coat protein (CP) gene、Nos terminator序列各設計專一性的引子對35S-726、CP-739、Nos-220，以總核酸抽取法抽取木瓜葉片核酸後，再以PCR增幅做快速檢定，訊號較弱的樣本可以再使用35S-348與CP-372做nested PCR以加強敏感度；也完成35S-726與Nos-220的multiplex PCR之研發，可有效的節省時間與成本；另又於35S promoter、CP、Nos terminator基因之間設計跨基因的引子對SC-670與CN-716，增加偵測的專一性與正確度；於Right boarder、Nos promoter、NPTⅡgene區域設計RBNP-308與NPTⅡ-718，可擴及應用到其他的基因轉殖作物之偵測。基因轉殖木瓜各個部位，不論根部、葉柄、第一片葉、完全展開葉、花、果實都可進行PCR檢測，市場果品的抽查以採集果皮的效果為佳。二、墨點雜合法（Dot blot hybridization）：此法主要是為了防止因遺傳變異，造成轉殖基因的部分片段漏失或突變而建立；為增強雜合訊號，選擇採用冷光（如CSPD）來反應，並可搭配AFLP（Amplification fragment length Polymorphism）之非專一性增幅，以提升偵測敏感度。三、Real-time PCR檢測法：以先前研發的PRSV TaqMan primer/probe方法為基礎，基因轉殖木瓜大約於第25個增幅循環即可增測到螢光訊號，可大幅減少檢測時間且可同步定量以利分析。將所研發的方法直接應用於田間基因轉殖木瓜之鑑定：在南台灣與東台灣田間調查可以發現，抗木瓜輪點病毒的基因轉殖木瓜在田間已有種植，且其中有部份也已經受病毒危害而產生病徵，經病毒檢測發現為木瓜畸葉嵌紋病毒（Papaya leaf-distortion mosaic virus）與PRSV的危害；中國廣西與海南島所採集到的木瓜樣本更發現基因轉殖木瓜已經大規模種植，且也有部份受PRSV入侵。將基因轉殖木瓜分別接種PRSV中國廣西分離株與台灣之PRSV-DF與PRSV-SMN系統，只有廣西分離株可以成功感染，而台灣系統則否。為瞭解廣西分離株之分子特性，取其鞘蛋白基因進行選殖與定序，再與國內外所發表的各PRSV鞘蛋白基因做比對，發現基因轉殖木瓜上所帶的PRSV鞘蛋白核酸序列和台灣PRSV永康分離株完全一樣、與中國廣西分離株相似度為94.1 %、與台灣DF、SM、SMN系統的相似度為96.3~96.5 %、與夏威夷與墨西哥分離株相似度為90.8 %與89.6 %；在胺基酸序列的親緣關係研究發現廣西分離株似乎和已知國內外系統沒有太大的差異。

This thesis was dedicated to develop various efficient methods with the DNA-based techniques for the identification of transgenic papayas resistant to PRSV. These developed methods contain (1) PCR detection：This was developed for detecting 35S promoter, PRSV coat protein gene, Nos terminator in transgenic papayas using our devised 35S-726, CP-739 and Nos-220 primer pairs. Nested-PCR assays were conducted using the 35S-348 and CP-372 primers to increase sensitivity. Both 35S-726 and Nos-220 primer pairs were used to perform multiplex PCR assays. Primer SC-670 could amplify the fragments between 35S promoter and PRSV CP gene；Primer CN-716 could amplify the fragments between PRSV CP gene and Nos terminator. Both SC-670 and CN-716 provide a more accurate and specific detection for the transgenic papayas. Primer RBNP-308 and NPTⅡ-718 designed to detect the right boarder region; Nos terminator and NPTⅡ gene were potential to be applied in the detection of other transgenic crops. All samples collected from young leaves, mature leaves, petioles, roots, flowers and fruits could be used in the detection of transgenic papayas. (2) Dot blot hybridization method: Dot blot hybridization provides a more accurate detection especially when gene-variation occurs in the inserted genes of transgenic papayas. AFLP techniques and fluoremetrical detection were used to increase sensitivity for dot blot hybridization in this study. (3) Real-time PCR method: This was applied for rapid detection and quantification of target gene for transgenic papayas. Our devised methods were directly applied in field survey for papaya plants. Many transgenic papaya plants were detected in the southern and eastern Taiwan. Interestingly, those transgenic papayas were also infected by PRSV in addition to PLDMV (Papaya leaf-distortion mosaic virus). Lots of papaya samples collected from Guangxi and Hainan (China) also showed positive for transgenic plants. It indicates that transgenic papayas have become the main cultivars in China. A PRSV isolate collected from Guangxi could infect transgenic papayas and caused typical symptoms whereas PSRV-DF and PRSV-SMN strains (from Taiwan) can not infect them. Molecular alignment and analysis of the coat protein gene revealed that the Guangxi isolate is somewhat different from the other reported PRSV isolates. Alignment of nucleic acid sequence demonstrated that this Guangxi isolate is 94.1% identical to the coat protein gene of transgenic papayas, and it is 96.3~96.5 % identical to the coat protein gene of PSRV-DF and PRSV-SMN strains. However, alignment of amino acid sequence did not show significant differences among them.