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

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。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.口試委員會審定書.........................................Ⅰ辭.....................................................Ⅱ文摘要.................................................Ⅲ文摘要.................................................Ⅳ錄.....................................................Ⅴ目錄...................................................Ⅷ目錄...................................................Ⅹ一章 前言...............................................1二章 前人研究...........................................2一節 園藝作物之採後生理變化.............................2、更年型與非更年型果實之介紹............................2、調控番茄果實後熟之相關基因表現........................3一) 乙烯生合成相關基因...................................3二) 小分子熱休克蛋白基因.................................5三) 茄紅素生合成相關基因.................................6二節 基因改造生物之概說................................13、什麼是基因改造生物...................................13、基因改造生物的運用與功能.............................14、基因改造作物之現況...................................16、基因改造食品安全性...................................17、各國基因改造產品規範.................................19三節 基因改造番茄之介紹................................35、番茄簡介.............................................35、不同特徵之基因改造番茄...............................36一) 延遲番茄果實後熟....................................36二) 具抗病蟲害能力之基改番茄............................38三) 具抗環境逆境之基改番茄..............................40四) 增加其營養價值之基改番茄............................42四節 基因轉殖植物之分子檢測技術........................45、以DNA為主之分子檢測技術 ..............................45一) 聚合酶鏈反應........................................45二) 南方轉漬分析........................................46、以mRNA為主之分子檢測技術.............................47一) 北方轉漬分析........................................47、以蛋白質為主之分子檢測技術...........................47一) 西方轉漬分析........................................48二) 二維膠體電泳........................................48三章 材料與方法........................................50一節 研究材料..........................................50二節 藥品..............................................50三節 儀器設備..........................................51四節 實驗方法..........................................52、以番茄DNA為主之分子檢測方式..........................52一) 基因改造與非基改之番茄果實DNA製備與純化.............52二) 南方轉漬分析........................................52三) 即時定量聚合酶鏈反應................................54、以番茄mRNA為主之分子檢測方式.........................55一) 收集番茄果實後熟相關之基因mRNA序列..................55二) 基因改造與非基改之番茄果實mRNA製備與純化............55三) 逆轉錄聚合酶連鎖反應................................56四) 即時定量聚合酶連鎖反應..............................56、以番茄蛋白質為主之分子檢測方式.......................57一) 基因改造與非基改番茄果實總蛋白質製備與純化..........57二) 重組嵌蛋白之大腸桿菌活化與表現......................57三) SDS-PAGE............................................58四) 西方轉漬分析........................................58五) 二維電泳分析........................................59四章 結果與討論........................................64一節 基因改造與非基改番茄之DNA分析.....................64、南方轉漬分析.........................................64、以即時定量聚合酶鏈反應確定轉入之鞘蛋白基因拷貝數.....65一) 定量標準曲線之建立..................................65二) 基改番茄果實中胡瓜嵌紋病毒鞘蛋白基因之拷貝數分析....66二節 基因改造與非基改番茄之mRNA分析....................74、以即時聚合酶鏈反應定量各後熟相關基因之表現...........74一) 以RIN-F/RIN-R引子對定量番茄後熟基因RIN表現..........74二) 以ACS2-F/ACS2-R引子對定量番茄後熟基因ACS2表現.......75三) 以ACO4-F/ACO4-R引子對定量番茄後熟基因ACO4表現.......75四) 以NR-F/NR-R引子對定量番茄後熟基因NR表現.............76五) 以PSY1-F/PSY1-R引子對定量番茄後熟基因PSY1表現.......76六) 以sHSP21-F/sHSP21-R引子對定量番茄後熟基因sHSP21表現.77七) 以vis1-F/vis1-R引子對定量番茄後熟基因vis1表現.......77、以即時聚合酶鏈反應定量胡瓜嵌紋病毒鞘蛋白基因之表現...78一) 以CPF/CPR引子對定量胡瓜嵌紋病毒鞘蛋白基因表現.......78三節 基因改造與非基改番茄之蛋白質分析..................100、以三種蛋白質萃取方式進行基因改造與非基改番茄之總蛋白質萃取...............................................100、以西方轉漬分析胡瓜嵌紋病毒鞘蛋白於基改番茄果實後熟過程中量之變化............................................100、以二維電泳分析基因改造與非基改番茄果實表現之總蛋白質差異性...............................................100五章 結論.............................................115考文獻................................................11