基因改造抗胡瓜嵌紋病毒番茄安全性與非預期效應評估之研究

Abstract

隨著基改作物的栽種生產日趨普及，以歐盟為首之諸多國家，對於基改作物的安全性與管制也越來越重視。本研究以亞洲蔬菜發展中心所研發出的國產基改抗胡瓜嵌紋病毒番茄R8為材料，進行安全性評估以及非預期效應的研究。本研究於第二章首先遵循衛生署所公布「基因改造食品」之安全評估方法進行過敏性評估。綜合本研究以及其他評估研究計畫的結果，雖然在農藝性狀與果實總糖含量上有顯著變化，但是沒有實際的安全性風險。第三章進行品系專一性即時定量聚合酶鏈反應檢測系統的建立，成功地建立具有足夠專一性、定量線性與靈敏度的檢測方法。此外藉由模擬胃液的分解，證實基改番茄R8 的果實DNA 並未有殘留於消化道的疑慮。第四章進行種植基改番茄R8 對於土壤微生物菌相的影響評估，以瞭解種植基改番茄R8 是否會對於土壤微生物生態帶來衝擊。結果顯示相較於試驗田中的其他環境因子，種植基改番茄R8對於土壤微生物不會顯著的衝擊土壤微生物生態。本研究的最後一部份為整合cDNA 微陣列分析與蛋白質二維電泳以及反轉錄-即時定量聚合酶鏈反應技術進行基改番茄R8 的非預期效應評估。結果顯示基改番茄R8 在轉殖過程中的確產生了非預期的改變，對於植株的生理發育以及果實的糖度可能產生了影響，但是並沒有發現具有安全性疑慮的非預期改變。在涵蓋性足以將所有的代謝變化進行描述的研究方法出現之前，整合多個分子層次的非預期效應研究方法有其必要性。綜合本研究結果，國產基改抗胡瓜嵌紋病毒番茄R8雖然基改番茄R8在農藝性狀、基因表現與蛋白質表現上與非基改番茄L4783有部分具有顯著差異，但是這些改變並未發現明顯的安全性的疑慮。過敏性與毒理學的評估的結果也顯示基改番茄R8 可以安全食用。近年來高通量分析技術的快速進步，以及生物分子資料庫的快速成長，使得有關植物生理代謝機制知識的快速進展。越來越深入的分子層次研究結果顯示出現行基於「實質等同」的法則所提供的資訊有可能不足以完全評估基改作物的安全性。雖然目前非預期效應的評估仍因為成本偏高及知識和技術的限制無法普遍實行，評估的結果也往往無法有清楚而完整的解釋，但對於基改作物的安全性評估來說，仍然具有相當的價值。如果現行基改作物安全性評估方法之外，加入非預期效應的評估，雖然可能無法立即清楚的判定基改作物的安全性，但隨著分析技術以及相關知識的快速進步，非預期效應評估的資料將可提升安全性評估決策的可靠性。

Following the widespread of genetically modified (GM) crop in the world, many countries such as EU have great concern about GM crop regulation and safety. A transgenic CMV-resistant tomato line R8 which developed by Asian Vegetable Research and Development Center was used to study the safety assessment and unintended effect of GM crop in this research. We start with the conventional safety assessment of GM crop in the chapter 2, and the GM tomato R8 was concluded as safe following the guideline of safety assessment. In the chapter 3 of this research, a real-time PCR detection system of GM tomato R8 was established with feasible specificity, linearity and sensitivity. The digestive fate of tomato fruit DNA was also revealed by simulated gastric fluid (SGF) digestion and real-time polymerase chain reaction analysis. Rapid degradation of tomato fruit DNA in the SGF showed that there is a minimal risk of gene flow. The chapter 4 of this study is the evaluation of the effect of GM tomato on soil microbial communities using denaturing gradient gel electrophoresis. The result of soil microbial analysis showed that the effect of GM tomato R8 was minor than the environmental factors such as soil position. Thus we conclude that GM tomato R8 has no detrimental impact on soil microbial communities. The last part of this study is the evaluation of the unintended effect of GM tomato R8 with proteomic and transcriptomic analysis. The results of this study have revealed the unintended changes of gene and protein expression in GM tomato R8, which may caused the significant changes of floral development and sugar content of GM tomato R8. In summary, GM tomato R8 did have several unintended changes in the gene and protein expression which may relate to the significant changes of floral development and sugar content. However, these unintended changes do not relate to any significant safety risk. The results of allergenicity and toxicity assessment were also showed no significant safety concern. Due to the complex and uncertain process of plant gene transformation, unintended changes GM crop should always exist. As the rapid development of high-throughput analysis, the unintended effects of GM crops have caused the contradiction of “substantial equivalence” concept used in the GM crop safety assessment. We think that the evaluation of unintended effects should combine with the “substantial equivalence” in the GM crop safety assessment in order to improve reliability of the decision making process in the future.