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|關鍵字:||食品科技, 生物技術;應用研究;transgenic papaya;基因改造基因木瓜;生物安全;實質等同;bio-safety;substantial equivalence||摘要:||
Based on theplicy for the establishing of transgenic food risk assessment plarform announced by the Department of Health, our collaborative research proups will use the transgenic papaya of Chung-Hsing University as materoals for establishment of both the transgenic food safety assessment of transgenic papaya detection system. The project include three sub-projects: (1) Stable sample supply for food biosafety assessment of transgenic papaya. (2) non-specific immune responses of domestic transgenic papaya TPY10-4 and allergenicity assessment on backcross breeding. (3) Levels of nutrients and anti-nutrients in transgenic papaya TPY10-4 and single-resistnat hohmzygotic parental lines. Single resistant to Papaya ringspot virus(PRSV)and double resistant to PRSV and Papaya leaf-distortion mosaic virus (PLDMV)transgenic papaya lines have been successfully developed by the Molecular Plant Virology Laboratory of National Chung Hsing University (NCHU). In this project, the selected single- and double-resistance transgenic papaya lines will be microprogagated by tissue culture. The main objective of this research project is supply the single resistant to PRSV transgenic fruits of Thailand and Sunrise varieties at different time for the experimental needs of food bio-safety assays in 2011. The transgenic Thailand and Sunrise varieties were obtained from several times of backcrossing transgenic line 1824 with parent lines. Total DNA extracted from leaves, peel and pulp of transgenic papaya and non-transformed control was used for variety identification by PCR with transgenic variety-specific primers. According to PCR results, test materials obtained from transgenic line was confirmed again. Non- transformed variety Tailand were determined with their horticultural traits. The test samples were divided into fresh and freeze-dried materials. Fresh fruit materials were collected depended on the experiments. Freeze-dried materials were prepared by professional manufacturer follows fruit characters analysis. In the first year (2010) the domestic transgenic papaya TPY10-4 will be used as the experimental materials to conduct the allergenicity assessment on non-specific immune responses using BALB/c mouse model in this study. The experimental design concerning allergenicity of transgenic papaya TPY10-4 using a non-specific immune response animal model is based on immunologic theory. The effects of test samples on the Th1/Th2 cytokine secretions by spleen cells, and the serum titers of non-specific antibodies in vivo will be determined. According to the results of the productions of Th1/Th2 cytokine (such as IFN-γ/IL-4 ratio) by immune cells, and the level changes in non-specific antibodies in vivo, especially IgE, compared to the non-transgenic papaya, the allergenic possibility of test samples will be judged. To further confirm the backcross stability of transgenic papaya, in the second year (2011) the papaya fruits from backcross breeding of domestic transgenic papaya resistant to PRSV coded as 1824 will be subjected to the allergenic assessment under specific immune responses using female BALB/c mouse animal model. As this study is finished, the backcross stability of transgenic papaya concerning allergenicity will be determined.In this sub-project (3), we investigated the chemical composition variances between the two (823 and 2210) cross anti-Papaya ringspot virus (PRSV) genetically modified (GM) and non-GM papaya fruits (including unripe and ripe); and the substantial equivalence assessment for two GM ones. The general analytic methods, high performance liquid chromatography, gas chromatography, and inductively coupled plasma emission spectroscopy along with two assessment methods (comparisons with the control and the international databases) were used. Of the 25 test parameters, the substantial equivalence rates for the unripe and ripe anti-PRSV GM 823 papaya fruits were 71 and 76%, respectively, as well as 76 % of rate for both unripe and ripe 2210 samples. The substantial equivalent results were not fully satisfactory, which were partly due to the sampling errors. Some constituent (i.e. fiber and vitamin C) levels in the papaya fruits were highly correlation with their ripe degree. When the unripe/ripe sampling standards were not consistent possibly resulting in the substantial equivalent assessment mistakes.
本計畫依據衛生署「建立我國基因改造產品風險評估環境建制計畫」要點，針對中興大學研發之轉基因木瓜提出此整合計畫。內容包過三項子計畫：一、供食品安全評估國產轉基因木瓜穩定樣品之提供；二、國產轉基因雙抗病毒木瓜TPY10-4之非特異性免疫及單抗病毒木瓜1824回交育種之過敏性評估；三、國產轉基因雙抗病毒木瓜TYP-104及回交育種之營養成分及抗營養成分分析。由中興大學植物病理學系分子植物病毒學研究室利用遺傳工程基因轉殖技術，成功育成單抗輪點病毒(Papaya ringspot virus; PRSV)及雙抗輪點病毒與畸葉嵌紋病毒(Papaya leaf-distortion mosaic virus; PLDMV )轉基因木瓜。本年度進行單抗輪點病毒回交親本-日昇及泰國品種木瓜之供果試驗。定植後及採果期間利用針對雙抗轉基因木瓜的外源基因設計專一性引子，抽取木瓜葉片、果皮及果肉之總量DNA，進行PCR，電泳分析結果確認各供試轉基因木瓜株系材料正確無誤，非轉基因對照則根據其園藝性狀判別。供試樣品分為鮮果材料和冷凍乾燥材料二種，鮮果材料視實驗需要不定期採集。冷凍乾燥材料採集後經過去皮及切片處理後，先初步進行果實各項性狀分析，冷凍乾燥後，交由其他子計畫進行試驗。本研究第一年(99年)將以中興大學育成之雙抗輪點病毒與畸葉嵌紋病毒基因轉殖木瓜TPY10-4為材料，進行非特異性免疫反應下之過敏性分析，以動物體內實驗探討輪點病毒及雙抗輪點病毒轉殖木瓜前後，木瓜對Th1/Th2細胞激素及非特異性抗體含量之影響，並依免疫指標之變化，判斷輪點病毒與畸葉嵌紋病毒基因轉殖後是否增加其過敏性。為探討基因改造木瓜之回交育種穩定性，本研究第二年(100年)將進行單抗病毒木瓜1824回交育種之過敏性評估，實驗將針對基因改造木瓜回交育種後的木瓜果實，進行特異性免疫評估，以了解基因改造木瓜回交育種後，過敏性是否發生變化。子計畫三分析二個回交基改抗木瓜輪點病毒之木瓜果果實 (青果、熟果) 於化學組成分之差異，並分析與未轉基因木瓜間『實質等同』之符合度。分析方法包括一般化學分析法、比色法、高效能液相層析法及感應耦合電漿發散光譜法等；實質等同評估方法包括與對照組及國際重要資料庫間之差異。期中報告之結果顯示出，以對照組評估法 (assessment with control) 聯合OECD參考數據評估法 (assessment with OECD data) 之結果，在分析之25個項目中，823青果及熟果之實質等同率均分別為71及76%；2210則均為76 %。二個回交基改木瓜之實質等同率均未達理想，推測其部分原因，係由於木瓜樣品採樣誤差，青果及熟果採摘之判斷基準不一；而一些組成分 (纖維素、維生素C等) 之含量與其成熟度有高度之關聯性，因此造成實質等同評估結果的偏差。
|Appears in Collections:||食品暨應用生物科技學系|
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