採後生理變化對抗胡瓜嵌紋病毒基改番茄之影響與安全性評估

Effect and safety assessment of the post-harvest physiology change of the genetically modified CMV-resistant tomato

巫佳霖(Chia-Lin Wu)

指導教授 ： 徐源泰

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02055

摘要

自1996年開始正式商業栽培基改作物起，引發全球消費者對基改作物與其衍生食品安全上之疑慮。基因改造作物之食品安全性評估主要採用「實質等同」(substantial equivalence)為基本原則，其以傳統食品為一般認定安全的(generally recognized as safe, GRAS)為出發點，將基改食品與傳統食品進行比較，若結果顯示無顯著之差異，則基改作物與其食品即可被視為安全的。故本試驗之目的為監測基因改造番茄於後熟過程中基因表達與其表現產物之可能變化，以確保安全性評估之完整性。以分子檢測之方式對基改番茄之DNA、RNA及蛋白質層面進行偵測。利用南方轉漬法與即時定量聚合酶鏈反應證實此基改番茄中轉入之胡瓜嵌紋病毒鞘蛋白基因於基因組中為單一拷貝數。再以即時定量聚合酶鏈反應分析基改與非基改番茄果實中七個與後熟相關基因之表現(RIN、ACS2、ACO4、NR、PSY1、sHSP21、vis1)，以及分析不同後熟程度之基改番茄果實中胡瓜嵌紋病毒鞘蛋白基因表現情形，同一後熟程度下，基改與非基改番茄果實中此七個後熟相關基因之表現量無顯著之差異；而抗胡瓜嵌紋病毒之基改番茄中，鞘蛋白基因表現量亦不隨基改番茄果實後熟之進行而使其基因表現量有所改變。以西方轉漬法分析基改番茄果實中胡瓜嵌紋病毒鞘蛋白之累積，結果顯示，此基改番茄果實所累積之鞘蛋白非常微量，可能無法以現有之技術偵測，故以呈色之方式進行西方轉漬訊號之偵測測試，得其偵測極限為0.1 μg的胡瓜嵌紋病毒鞘蛋白，代表此基改番茄果實與葉片中胡瓜嵌紋病毒鞘蛋白之累積量低於總蛋白質的0.05%，此轉殖胡瓜嵌紋病毒鞘蛋白基因之基改番茄果實與葉片中無大量表現病毒之鞘蛋白；再將胡瓜嵌紋病毒鞘蛋白基因之表現量與不同後熟階段下之後熟相關基因表現量進行比較，證實鞘蛋白基因之表現量較其他七個後熟相關基因表現量低且達顯著性差異，故推測︰此基改番茄株具抗胡瓜嵌紋病毒病害之能力可能藉後轉錄基因靜默原理。目前利用二維電泳監測抗胡瓜嵌紋病毒之基改番茄與其母本番茄株，觀察兩者果實中總蛋白質之差異表現情形，兩者果實中總蛋白質之表現情形具差異性，可能因環境因素與人為操作上之誤差，以致兩者之蛋白質差異表現，故尚須利用西方轉漬法與質譜儀分析技術針對具差異性表現之蛋白質做進一步之身份確認。

關鍵字

胡瓜嵌紋病毒 ； 基因表現差異 ； 基因改造番茄 ； 採後生理變化 ； 即時定量聚合酶鏈反應 ； 西方轉漬分析

並列摘要

Since the commercialization of first transgenic crop in 1996, safety of genetically-modified crops and its related products become major concerns of consumer. Currently, environmental risk assessment brings by transgenic crops and food safety are the two main issues worldwide. To meet global regulatory authorities on the practice of substantial equivalence on transgenic food, which means that food derived from crops produced through biotechnology be as safe as food produced from conventionally bred crops, this study has investigated the variations of the gene expressions and protein accumulations in transgenic tomato fruits throughout the post-harvest and processing processes. We used Southern blotting and real-time PCR to verify single copy of CMV coat protein gene found within the genome of the transgenic tomato lines in this experiment. Comparison of seven ripening-related gene expressions between genetically modified (GM) and non-GM tomato fruits at different ripening stages by real-time quantitative PCR were also carried out. No significant differences were found in seven ripening-related gene expressions among GM and Non-GM tomato fruits at the same ripening stage. Furthermore, the expression of exogenous gene - CMV coat protein showed no significant changes in GM tomato fruit among different ripening stages. Detection of CMV coat protein among different ripening stages of transgenic tomato fruits is done by Western blotting. The CMV coat protein accumulations is extremely low in transgenic tomato fruits. The limitation of colorimetric detection was about 0.1 μg of purified CMV coat protein. We further compared with exogenous gene and ripening-related gene expressions among GM tomato fruits. The CMV coat protein gene transcripts were significantly lower than ripening-related gene transcripts. Therefore, it is presumed that post-transcriptional gene silencing mechanism may be the reason of anti-virus ability in transgenic tomato. Subsequently, two-dimensional electrophoresis was used to analysis the differential accumulations of total proteins among GM and Non-GM tomato fruits. The results indicated that there were some differential accumulations of proteins between GM and Non-GM tomato fruits. However the differential expression of proteins may be due to the environmental changes or experiment manipulation errors. Therefore, further study should focus on the properties of the differential proteins using western blotting or mass spectrometry.

並列關鍵字

cucumber mosaic virus ； differential gene expression ； genetically modified tomato ； post-harvest physiology change ； real-time PCR ； western blotting

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