|Title:||基因轉殖植物檢測技術開發體系之建立||Other Titles:||The proposed protocol for the detection system of transgenic plants in Taiwan||Authors:||范明仁
|Keywords:||基因轉殖;木瓜輪點病毒;普魯南醣酶;乳鐵蛋白;植酸酵素;Gene transformation;Papaya ringspot virus (PRSV);amylopullulanase (APU);Lactoferrin;Phytase||Issue Date:||Dec-2005||Publisher:||農業試驗所||Related Publication(s):||農業試驗所特刊第120號||Start page/Pages:||105-129||Source:||基因轉殖植物之生物安全評估與檢測專刊||Conference:||基因轉殖植物之生物安全評估與檢測研討會||Abstract:||
基因轉殖植物之檢測有兩種方式，第一種是藉由基因表現的層次進行觀察檢測，如：基因表現蛋白質或植物之外在表徵等；第二種方式係就DNA 層次直接檢測轉殖之外來基因的DNA 序列。就基因表現層次方面而言，目前台南區農業改良場利用除草劑進行耐藥性篩檢，供試的21 個玉米品種均對嘉磷塞(Glyphosate)（年年春41%S、80 倍稀釋液噴施）及固殺草(Glufosinate)（百試達18.02%S、100 倍稀釋液噴施）無耐藥性。僅進口飼料玉米具有耐藥性之個體。花蓮區農業改良場則已發展以x-gluc 溶液染色法檢測具GUS 基因之基因轉殖植物之技術。再就DNA 層次之檢測方式而言，其可區分為四個檢測層次，第一層次為初步篩檢是否為基因轉殖作物，就木瓜而言，農業試驗所針對CaMV35S 啟動子（Cauliflower mosaic virus 35S promoter；CaMV35S）、nos（nitric oxide synthase；nos）終結子，以及抗Kanamycin 之篩選基因nptII（neomycin phosphotransferase II；nptII）；高雄區農業改良場針對CaMV35S 啟動子及nos終結子進行檢測，皆可獲得專一性條帶，而非基因轉殖木瓜無此條帶；台南區農業改良場則是針對玉米及番茄進行CaMV35S 啟動子及nos 終結子檢測是否有基因轉殖作物混雜，結果顯示21 個玉米品種(主要為甜玉米)及42 個番茄品種均未檢測出有基因轉殖植物滲入，僅進口飼料玉米檢測出。第二層次為標第基因特異性檢測，農業試驗所針對PRSV（papaya ringspot virus）鞘蛋白結構基因序列所設計之引子，可使16-0-1、17-0-5、18-2-4 等基因轉殖木瓜株系產生約820bp 之專一性條帶，非基因轉殖木瓜無此條帶。第三層次為檢測外源基因之結構特異性，引子所擴增區域係跨越在二個以上的外源基因，藉此方式可順利判別基因轉殖之外源基因間相對位置，農業試驗所以此方式，已順利獲得轉殖木瓜外源基因之完整序列，可明確區分特定轉殖與非轉基因轉殖木瓜。第四層次為事件特異性檢測，藉由各轉殖株系間，外源基因插入點之差異，區別不同之轉植株系。農業試驗所已成功設計不同引子，對不同木瓜轉殖株系有專一性的判別條帶。另外於水稻方面，目前農業試驗所已建立水稻基因轉殖鑑定技術結果顯示，第一層次以PCR 方式可偵測出轉殖水稻含有CaMV35S 啟動子、nos 終結子，以及篩選基因hptII（hygromycin resistance；hptII）之專一性條帶，非基因轉殖水稻無此條帶；第二層偵測出基因轉殖水稻分別含有APU（amylopullulanase）、Lactoferrin、Phytase 等基因；第三層次針對基因轉殖APU，以及轉殖Phytase水稻進行外源基因跨區檢測，亦可獲得專一的條帶，經定序後與原始已知序列相同。
Generally there are two kind methods to detect transgenic plants. The first method is the detection of the character protein produced by gene translation from transgenic plant. Secondary method is DNA sequence detection of foreign gene transfer from other organisms. Tainan District Agriculture Research and Extension station (Tainan-DARE) had been survey some corn varieties for herbicide resistance. From their report, twenty-one traditional corns are not anti-herbicide by glyphosate (80X, 41%S glyphosate) and glufosinate (100X, 18.02%S glyphosate). Only imported feed corn has herbicide resistance of glyphosate and glufosinate. The detection method of transgenic plants in Hualien DAIS is to use x-gluc staining for those plants with GUS gene.
For the secondary detection method, which use DNA sequence as detection methods, can be classified into four categories. The first category is to assort transgenic plant and non-transgenic plants. For category 1 of identification, the primer sets of Taiwan Agriculture Research Institute (TARI) are designed to detect the CaMV35S promoter gene, nos terminator gene and the kanamycin selection marker gene nptII for papaya. Kaonsiung DARE use primers to detect the CaMV35S promoter gene and nos terminator gene, too. The results showed the transgenic papaya have specific bands; all of the traditional varieties don’t have those bands. The primer sets of Tainan DARE are designed to detect the CaMV35S promoter gene and nos terminator gene for corn and tomato. The results showed both twenty-one traditional corn varieties and forty-two tomato varieties don’t have specific bands, but imported feed corn has some specific bands. The category 2 was called as gene specific detection, TARI use the coat protein gene sequence of papaya ring spot virus (PRSV) from professor Yeh to design a primer set which could detect PRSV transgenic papaya by the specific product in 820bp, all of the traditional varieties don’t have those bands. A primer set of category 3 was designed for construct specific detection to amplify the region across between gene and gene. The results show accurate complete sequence from T-DNA of transgenic papaya by TARI. The category 4 was called as event specific. The T-DNA random inserts genomic DNA of every one transgenic plant line in different site. The primer sets of TARI are designed to detect the DNA flanking in papaya. The results showed papaya had specific bands in different transgenic lines. TARI also has accomplished the detection methods for detection transgenic rice. The PCR (Polymerase Chin Reaction) analysis is applied in this research. In category 1, the results showed transgenic rice have specific bands of CaMV35S promoter gene, nos terminator gene and the hygromycin selection marker gene hptII. All of the traditional varieties don’t have those bands. In category 2, the results showed transgenic rice have specific bands of amylopullulanans (APU) gene, lactoferrin gene and phytase gene. A primer set of category 3 was designed for construct specific detection to amplify the region across between gene and gene. The results showed accurate complete sequence from T-DNA of transgenic rice of APU and phytase.
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