無篩選標誌基因之葉綠體基因轉殖系統的應用

Resistance to antibiotics and herbicides mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, the presence of antibiotic and herbicide resistance genes in genetically engineered crops has generated a number of environmental and consumer's concerns for long time. Expression of foreign genes via chloroplast genomes offers several unique advantages, including high protein levels, expressing multiple genes in operons, transgene containment by maternal inheritance, as well as lack of gene silencing, position and pleiotropic effects. The most widely used selectable marker for chloroplast transformation is a gene encoding aminoglycoside 3'-adenylyltransferase (aada). Transplastomic plantlets are then selected by spectinomycin and/or streptomycin resistance. However, there are concerns associated with antibiotic resistance marker genes including the metabolic burden imposed by high-level expression of chloroplast and the potential for development of resistant strains of bacteria. D-amino acid racemase (alaR) can mediate the racemization of D-amino acids. D-amino acid oxidase (DAAO) catalyzes the dehydrogenation of D-amino acids to yield α-keto acid and accompany with NH3 and H2O2 production. Our previous studies has successfully employed biolistic bombardment to obtain a stable and highly efficient transformation platform for transplastomic cabbageand rice by using the alaR or daao gene as a selectable marker gene, conferring resistance to the D-alanine. Moreover, we had demonstrated the elimination of marker gene by the mechanism of transient cointegration of the marker gene. By using the system of daao gene as marker genes and followed by elimination of marker gene, we propose to engineering the phage lysozyme (lys) and holin (hol) genes into the chloroplast of cabbage, pai-tsai and rice. The objective of this study is to engineer transplastomic cabbage, pai-tsai and rice with a high level of resistance against plant disease using the approach of the marker gene-free technology.隨著基因改造作物的廣泛種植，其對人類建康及環境生態的衝擊，也廣受到社會大眾關著。目前被廣泛應用於轉殖作物篩選上的耐抗生素及抗除草劑標誌基因，將面臨減少或無法使用。植物葉綠體基因轉殖具有：增加轉殖基因大量表現，不會造成基因污染、基因靜寂及插入位置效應，與較細胞核基因轉移穩定等優點；因此開發葉綠體基因轉殖技術為近代生物技術的主力研發工作。由於葉綠體基因的大量表現轉殖之基因，因此對非耐抗生素基因或無篩選標誌基因之轉殖系統的需求，更具必要性及急迫性。D型胺基酸氧化酵素(DAAO)及D-丙胺酸消旋酵素(alaR)可以代謝D型胺基酸。 本研究之先期試驗已建立以alaR或daao基因作為篩選標誌基因，用D-alanine篩選甘藍、小白菜及水稻之葉綠體基因轉殖系統，並證實剔除篩選標誌基因之無篩選標誌基因的葉綠體基因轉殖系統是可行的。本研究乃結合所建立的daao基因作為葉綠體篩選標誌基因系統，及剔除篩選標誌基因之無篩選標誌基因的葉綠體基因轉殖系統，應用於植物抗病基因工程的研發。本研究計畫擬將分離與構築之lysozyme (lys)及holin (hol)基因轉移至甘藍及水稻之葉綠體中，探討利用無篩選標誌基因之葉綠體基因轉殖系統，培育出抗病甘藍、小白菜及水稻之可行性。此外，利用lysozyme及holin基因表現抗病的特性，當篩選標誌基因剔除後，方便篩選基因轉殖植株後裔，健全無篩選標誌基因之葉綠體基因轉殖系之建

Using D-Amino Acid Oxidase Gene (daao) as the Selectable Marker Gene for Cabbage Chloroplast Transformation

在作物基因轉殖過程中，利用篩選標誌基因(selectable marker genes)可 以快速且有效率的區分出轉殖與非轉殖細胞。然而在獲得轉殖植株後，篩選標誌基因便無特殊用途，並可能產生環境生態以及生物安全性等方面的疑慮。因此，目前被廣泛應用於轉殖作物篩選上的耐抗生素及抗除草劑標誌基因，將面臨減少或無法使用的命運。植物葉綠體基因轉殖具有增加轉殖基因大量表現、不會造成基因污染、基因靜默及插入位置效應、及較細胞核基因轉移穩定等優點；因此開發葉綠體基因轉殖技術為近代生物技術的主力研發工作。由於葉綠體基因的大量表現轉殖之基因，因此對非抗抗生素基因的篩選標誌基因的需求，更具必要性及急迫性。 D型胺基酸氧化酵素(D-amino acid oxidase, DAAO)是一種以FAD做為輔基的黃素蛋白，可以催化D型胺基酸的脫氨氧化反應。由於大部份D型組態的胺基酸，不能被植物代謝，且易對植物產生毒害。因此本研究以甘藍為材料，建立利用daao基因作為葉綠體基因轉殖之篩選標誌基因的系統。 本研究是將以分離自三角酵母(Trigonopsis variabilis)中的daao基因及耐抗生素之aada基因為篩選標誌基因、並以egfp及gus為報導基因，所構築之甘藍葉綠體基因轉殖載體，藉由基因槍法轉殖至甘藍的葉綠體。本研究之目的為：(一)、探討以 daao 基因作為甘藍葉綠體基因轉殖的篩選標誌之可行性，(二)、比較daao基因及aada基因作為甘藍葉綠體基因轉殖的篩選標誌之優劣。 將已構築之pMT91-ED(egfp-daao)、pMT91-GD(gus-daao)、pMT91-EDA (egfp-daao-aada)、以及pMT91-GDA (gus-daao-aada)等四種載體，利用基因槍法將其轟擊至`初秋´甘藍之下胚軸或葉片的葉綠體。再生培殖體經200~500 ppm D-alanine或20~50 ppm spectinomycin持續篩選，可以獲得再生植株。轉殖植株葉片以PCR及RT-PCR分析之結果顯示，daao及aada篩選標誌基因及egfp及gus報導基因已存在於轉殖甘藍之基因組中，並表現其mRNA。GFP綠色螢光及GUS活性染色分析的結果也顯示egfp及gus等報導基因亦可在轉殖甘藍中順利表現綠色螢光及藍色反應。本研究結果顯示以daao基因來作為甘藍之葉綠體基因轉殖的篩選標誌基因是可行的。本研究已初步完成建立以D-alanine來篩選甘藍之葉綠體基因轉殖系統。由於甘藍培殖體對spectinomycin之忍受毒害濃度的高敏感性，因此發展D-alanine的篩選系統，在植株外觀的篩選比Spectinomycin篩選的優點更顯而易見。In the process of transgenic crops, the use of selectable marker genes can be quickly and efficiently distinguish transgenic and non-transgenic cells. However, the antibiotic and herbicide resistance marker genes have generated a number of environmental, ecological, and biological aspects of safety concerns. Therefore, the development of antibiotic-free selectable marker genes is of outstanding importance for commercialization of transgenic crops. Expression of foreign genes via plastid genomes not only dramatically enhances the level of expression and absence of epigenetic effects, but also prevents out cross of the introduced foreign genes via pollen grains. Thus, transformation of the plastid genome is a new and attractive alternative to engineering the nuclear genome. D-amino acid oxidase (DAAO) catalyzes the oxidative deamination of D-amino acids to produce the corresponding keto acids, NH3 and H2O2. Most of the D-configuration amino acids cannot be metabolized by plants, and deterioration the growth of plants. In this study, the possibility of using D-amino acid oxidase gene (daao) as an antibiotic-free selectable marker gene in cabbage plastid gene transformation is studied. The objectives of this study are to develop and establish the daao gene as the antibiotic-free selectable marker for cabbage plastid transformation, and to compare the transformation efficiency between using aada (aminoglycoside-3’- adenyl transferase) and daao genes as the selectable marker. A set of plasmid vectors that contain daao gene cloned from the yeast (Trigonopsis variabilis) as antibiotic-free selectable marker was constructed by Dr. M. T. Yang’s laboratory. One of the two plant reporter genes, egfp and gus, was chosen as target gene in the constructed vectors in this study. The selectable marker (daao, aada) and reporter genes (egfp, gus) were cloned as a cassette. All of the constructed genes were driven by the Prrn promoter and terminated by T-psbA terminator. Four cabbage plastid transformation vectors, pMT91-ED (egfp-daao), pMT91-GD (gus-daao), pMT91-EDA (egfp-daao-aada), and pMT91-GDA (gus-daao-aada) were transferred into the hypocotyls or leaves of cabbage chloroplast via particle gun mediated transformation. The regenerated plantlets were induced and selected by 200 to 500 ppm D-alanine or 20 to 50 ppm spectinomycin。 The results of PCR and RT-PCR analysis indicated that transformed genes (daao, aada, gus, and egfp) were integrated into the plastid genome of transplastomic cabbage plants, and expressed its mRNA. GFP fluorescence and gus histochemical staining analyses showed the emission of green fluorescence and blue-color reaction were presented in the egfp or gus gene transformed transplastomic cabbage plants, respectively. Our results indicated that the system of using daao gene as selection marker for plastid transformation, which has several advantages over the conventional used aada gene, offers new possibilities for non-antibiotics selectable marker in commercially important crops.中文摘要…………………………………………………………………………1 英文摘要…………………………………………………………………………2 前言…………………………………………………………………………3 前人研究…………………………………………………………………………5 ㄧ、植物基因轉殖之相關研究概況…………………………………………5 (一)、葉綠體轉殖系統之研究…………………………………………5 (二)、基因槍轉殖系統導入外源基因至葉綠體基因組之研究………9 (三)、甘藍(Brassica oleracea var. capitata)基因轉殖之研究………10 二、轉基因作物之標誌基因的應用及其對環境安全產生之衝擊………12 (一)、aada基因及其在篩選標誌基因上之應用……………………13 (二)、D型胺基酸氧化酵素及其在篩選標誌基因之相關研究………14 (三)、gus基因及其在植物基因轉殖上的應用………………………15 (四)、綠色螢光蛋白(GFP)的相關研究………………………………17 (五)、植物安全性篩選標誌基因之研究………………………………20 材料方法………………………………………………………………………22 結果……………………………………………………………………………31 ㄧ、轉殖載體之檢驗……………………………………………………31 二、 D-alanine及Spectinomycin對甘藍本葉及下胚軸培養再生之影響…32 三、甘藍葉綠體基因轉移之培殖體篩選及誘導植株再生情形…………33 四、轉殖甘藍植株之基因及表現分析……………………………………34 討論……………………………………………………………………………69 ㄧ、轉殖載體之檢驗………………………………………………………69 二、 D-alanine及Spectinomycin對甘藍本葉及下胚軸培養再生之影響…69 三、甘藍葉綠體基因轉移之培殖體篩選及誘導植株再生情形…………70 四、轉殖甘藍植株之基因及表現分析……………………………………71 五、結 論……………………………………………………………………73 參考文獻………………………………………………………………………7

Using D-Amino Acid Oxidase Gene (daao) as the Selectable Marker Gene for Cabbage (Brassica oleracea L. var. capitata L.) via Agrobacterium Mediated Transformation

隨著基因改造作物的廣泛種植，其對人類建康及環境生態的衝擊，也廣受到社會大眾關著。目前被廣泛應用於轉殖作物篩選上的抗抗生素及抗除草劑標誌基因，將面臨減少或無法使用，因此對非抗抗生素基因的篩選標誌基因的需求，是具必要性及急迫性。大部份D型組態的胺基酸，不能被植物代謝，且對植物產生毒害。D型胺基酸氧化酵素(D-amino acid oxidase, DAAO)可以催化D型胺基酸的脫氨氧化反應(oxidative deamination)。本研究探討利用daao基因作為轉殖植物篩選標誌基因之應用潛力。本研究之目的為：(一)、建立以 daao 基因作為甘藍之農桿菌基因轉殖法的篩選標誌基因系統，(二)、比較daao基因及nptII基因作為甘藍之農桿菌基因轉殖法的篩選標誌基因之優劣。 本研究已完成將篩選自三角酵母(Trigonopsis variabilis)中的daao基因及nptII基因為標誌基因，以gus及egfp為目標基因，利用農桿菌轉移法將pCaPDAO-gus、pCaPDAO-egfp、pRPDAO-gus、pRPDAO-egfp等質體之上述基因轉移到`初秋´甘藍下胚軸。經D-alanine 或kanamycin篩選並誘導、再生成植株。轉殖植株以PCR分析之結果顯示，daao及nptII等篩選標誌基因已存在於轉殖甘藍之基因組中。GFP綠色螢光及GUS活性染色分析的結果顯示egfp及gus等目標基因可在轉殖甘藍中分別表現綠色螢光及藍色反應。 本研究顯示D-alanine的篩選系統在農桿菌介導的甘藍轉殖是可行的，並已初步完成建立甘藍之D-alanine篩選系統。由於甘藍培殖體對kanamycin之忍受毒害濃度的高敏感性，因此發展D-alanine的篩選系統，在植株外觀的篩選比kanamycin篩選的優點更顯而易見。Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, the presence of antibiotic resistance genes in genetically engineered crops has generated a number of environmental and consumer concerns for long time. Most of the amino acids found in nature are of the L-type. Hence, eukaryotic proteins are always composed of L-amino acids although D-amino acids are found in bacterial cell walls and in some peptide antibiotics. Some D-amino acids were very toxic to plants (e.g., D-alanine and D-methionine), while others had slight negative effects, and still others had no effect at all. One of the best known enzymatic pathways for metabolism of D-amino acid is via oxidative deamination by D-amino acid oxidase (encoded by the daao gene). In this study, the possibilities of using D-amino acid oxidase genes as selection marker genes in cabbage transformation are studied. The objectives of this study are to establish the system of daao gene as the selectable marker for cabbage transformation via Agrobacterium, and to compare the efficiency of nptII (neomycin phosphotransferase II) and daao genes as the selectable marker. The daao cloned from the yeast (Trigonopsis variabilis), nptII, gus and egfp genes were transfer into the hypocotyl of cabbage via Agrobacterium mediated transformation. The regenerated plantlets were selected by D-alanine or kanamycin. The results of PCR analysis indicated that daao and nptII genes were presented in the genome of transformed cabbage plants. GFP fluorescence and Gus histochemical staining analyses showed the emission of green fluorescence and blue-color reaction were presented in the egfp or gus gene transformed cabbage plants, respectively. Our results indicated that the system of using daao gene as selection marker, which has several advantages over the conventional used nptII, offers new possibilities for non-antibiotics selectable marker in commercially important crops.中文摘要...1 英文摘要...2 前言...3 前人研究 一.轉基因作物之標誌基因在轉殖後的危害...5 二.植物安全性篩選標誌基因之研究...6 三.轉基因作物中標誌基因的惕除...9 四.D型胺基酸氧化酵素(D-amino acid oxidase)及其在標誌基因之研究...10 材料與方法...12 結果 一.轉殖載體之聚合酵素鏈鎖反應分析...17 二.D-型胺基酸對甘藍及阿拉伯芥種子發芽與生長之影響...17 三.pCaPDAO-gus、pCaPDAO-egfp、pRPDAO-gus、pRPDAO-egfp質體 轉殖到`初秋´甘藍...18 四.轉殖甘藍植株之基因及表現分析...20 討論 一.Kanamycin 及D-alanine應用在篩選轉殖甘藍...43 二.甘藍之再生過程...44 三.轉殖甘藍植株之基因及表現分析...45 參考文獻...46 附錄...5

Studies on Transformation of D-Amino Acid Oxidase (daao), Transglutaminase (tga), and Lysozyme (lys) Genes into Pak-choi (Brassica campestris L. ssp. chinensis (L.) Makino)

由於小白菜具有生長快速、高環境耐受性的優點，因此栽培容易，也較不受到氣候環境等自然條件的限制。小白菜具有重要的民生及經濟地位，是台灣可全年生產的重要葉菜類蔬菜之一。過去三十年來，雖然育種者在小白菜改良上卓有成效，但是受限於物種差異，傳統育種在少數幾種種原親本間能進行育種改良的部分已達極限，因此為了培育出更具有耐環境逆境與高品質的小白菜品種，提升小白菜的附加經濟價值與競爭力，利用基因轉殖技術培育新的小白菜品種是一很好的選項。 本論文第二章首先探討不同D型胺基酸的種類及濃度對甘藍、水稻、結球白菜、小白菜種子發芽與生長之影響。其目的為建立一個適當的D型胺基酸的種類及濃度，可有效的應用在daao篩選標誌基因，以建立一安全、完善的非抗生素藥劑(D型胺基酸)作為篩選轉殖植株的基因轉殖系統。本研究結果顯示20 mM的D-alanine、D-asparagine、D-methionine、D-lysine等4種不同D型胺基酸中，D-alanine及D-methionine強烈抑制甘藍、結球白菜與水稻的的發芽與生長，D-asparagine次之，D-lysine的抑制效果較少。完全抑制水稻、甘藍、結球白菜種子發芽與生長之4種D型胺基酸約為 20 mM D-alanine、40 mM D-methionine (甘藍80 mM D-methionine)、40~80 mM D-asparagine、125 mM D-lysine。D-alanine次致死濃度 (sub-lethal dose) 之研究顯示甘藍在5~10 mM、水稻在10~15 mM、小白菜在2.5~5 mM之間的D-alanine濃度有一明顯的抑制芽梢的生長，而後再提高D-alanine濃度即有嚴重抑制芽梢的生長與成活。本研究建議進行甘藍、水稻、小白菜轉殖培植體的初次(早期)篩選時，D-Ala濃度在次致死濃度之間，待轉殖培植體的組織狀態穩定後，再逐步提高D-Ala濃度，獲得再生轉殖植株的機率可大幅提昇。 本論文第三章探討利用daao基因作為轉殖植物篩選標誌基因之應用潛力。本研究之目的為：(一)、建立以 daao 基因作為甘藍、水稻與小白菜之農桿菌基因轉殖法的篩選標誌基因系統，(二)、建立以 daao 基因作為小白菜之基因槍基因轉殖法的篩選標誌基因系統。本研究已完成將篩選自三角酵母(Trigonopsis variabilis)中的daao基因及nptII (對照)基因為標誌基因，利用農桿菌轉移法將pCDAD及pRDAD等質體之上述基因轉移到甘藍、水稻與小白菜培植體。經D-alanine 或kanamycin篩選並誘導、再生成植株。轉殖植株以PCR分析之結果顯示，daao及nptII等選標誌基因已存在於轉殖甘藍、水稻與小白菜之基因組中。本研究同時將gus、daao及/或aadA基因，利用基因槍轉移法將pMT91-GD及pMT91-GDA等質體轉移到小白菜葉綠體中。經D-alanine 或spectinomycin篩選並誘導、再生成植株。轉殖植株以PCR及RT-PCR分析之結果顯示，gus、daao及aadA等基因已存在於轉殖T1及T2小白菜之基因組中，並表現daao mRNA。本研究顯示D-alanine的篩選系統在農桿菌介導法及基因槍轉移法的植物基因轉殖是可行的，並已初步完成建立小白菜之D-alanine篩選系統。由於小白菜培植體對spectinomycin及kanamycin之忍受毒害濃度的高敏感性，因此發展D-alanine的篩選系統，在植株外觀的篩選比spectinomycin及kanamycin篩選的優點更顯而易見。 本論文第四章以D型胺基酸氧化酵素(daao)及D-型胺基酸消旋酵素(D-amino acid racemase, alaR)基因作為篩選標誌基因，將轉穀氨醯胺酵素基因(tga)轉移至小白菜之葉綠體中。本研究之目的為探討利用小白菜葉綠體為非抗抗生素篩選標誌基因的生物反應器，生產轉穀氨醯胺酵素的可行性，以提高小白菜之經濟效益。本研究將自放線菌Streptomyces netropsis中篩選出的轉榖氨醯胺酵素基因(tga)，所構築到小白菜葉綠體基因轉殖之八種載體pMT91-GTA、pMT91-GPA、pMT91-ETA、pMT91-EPA、pMT91GPsDA、pMT91GPsRA、pMT91EPsDA和pMT91EPsRA，利用基因槍法轉殖到'台農3號'小白菜之本葉的葉綠體。培植體經100~300 mg/L D-alanine或1~30 mg/L spectinomycin漸進篩選及誘導再生，目前已獲得T3轉殖子代。T0~T3 葉片經PCR分析之結果顯示，tga基因已存在於轉殖小白菜之葉綠體基因組中，且部份alaR及aadA等篩選標誌基因已被惕除，不存在小白菜的葉綠體基因組中。轉殖小白菜之葉片可偵側到TGA酵素活性，其中有二個轉殖系之TGA酵素活性較未轉殖對照組增加150~200%。本研究之結果顯示以daao及alaR等非抗抗生素篩選標誌基因之葉綠體基因轉殖技術，應用在小白菜葉綠體轉殖植株作為生物反應器，以生產轉穀氨醯胺酵素是可行的。 本論文第五章之研究目的為探討利用無篩選標誌基因之葉綠體基因轉殖系統，培育出抗病小白菜之可行性。本研究將分離自Xanthomonas fragariae (草莓角斑病菌)菌株的類似噬菌體 (phage XF)的溶菌酶 (lys)，並構築到甘藍葉綠體基因轉殖之四種載體 (pMT91-GLsA、pMT91-ELsA、pMT91F-GLsA、pMT91F-ELsA)，利用基因槍法轉殖到'台農3號'小白菜之本葉的葉綠體。培植體經1~30 mg/L spectinomycin漸進篩選及誘導再生，目前已獲得T1轉殖子代。T0及T1葉片經PCR及RT-PCR分析之結果顯示，lys基因已存在於轉殖之小白菜的葉綠體基因組中，並可表現lys mRNA。本研究初步結果顯示，利用葉綠體基因轉殖系統，轉殖溶菌酶基因 (lys)到小白菜，是可行的。中文摘要…………………………………………………………………………………i 英文摘要………………………………………………………………………………iii 目次……………………………………………………………………………………vi 表目次…………………………………………………………………………………ix 圖目次…………………………………………………………………………………x 緒言……………………………………………………………………………………1 前人研究………………………………………………………………………………4 一、葉綠體基因之相關研究……………………………………………………4 二、非抗生素篩選基因之研究…………………………………………………7 (一)、菠菜甜菜鹼醛脫氫酶(badh)………………………………………… 7 (二)、D型胺基酸代謝基因(daao、alaR)……………………………………8 三、剔除篩選基因之研究………………………………………………………10 (一)、經由直接重複序列的同源重組切除篩選基因………………………10 (二)、藉噬菌體位置專一重組酶(phage site-specific recombinase)切除 篩選基因……………………………………………………………11 (三)、篩選基因的短暫存在以獲得無篩選基因轉殖株……………………12 (四)、共轉殖-隔離法(Cotransformation-segregation)以獲得無篩選基因 轉殖株………………………………………………………………13 四、葉綠體基因轉殖技術之應用………………………………………………13 (一).葉綠體基礎研究之進展……………………………………………13 (二)、改善植物抗性之研究…………………………………………………14 1、蘇力菌cry基因…………………………………………………14 2、β-葡萄糖苷酶基因bgl-1………………………………………………15 3、抗菌胜肽(antimicrobial peptide) ………………………………………16 4、溶菌酶(lysozyme) ………………………………………………………17 五、轉穀氨醯胺酵素應用………………………………………………………18 第二章、D型胺基酸對水稻、甘藍、結球白菜、小白菜種子發芽與生長之 影響…………………………………………………………………………19 摘要………………………………………………………………………………19 英文摘要…………………………………………………………………………20 前言………………………………………………………………………………21 材料與方法………………………………………………………………………23 結果………………………………………………………………………………25 討論………………………………………………………………………………39 第三章、建立daao基因作為甘藍、水稻、小白菜基因轉殖的篩選標誌基因之 研究…………………………………………………………………………42 摘要………………………………………………………………………………42 英文摘要…………………………………………………………………………43 前言………………………………………………………………………………45 材料與方法………………………………………………………………………48 結果………………………………………………………………………………57 討論………………………………………………………………………………78 第四章、轉殖tga基因到小白菜(Brassica campestris L. ssp. chinensis (L.) Makino) 葉綠體之研究………………………………………………………………82 摘要………………………………………………………………………………82 英文摘要…………………………………………………………………………83 前言………………………………………………………………………………84 材料與方法………………………………………………………………………87 結果……………………………………………………………………………95 討論……………………………………………………………………………114 第五章、轉殖溶菌酶(lysozyme)基因至小白菜(Brassica campestris L. ssp. chinensis (L.) Makino)葉綠體之研究……………………………………118 摘要……………………………………………………………………………118 英文摘要………………………………………………………………………119 前言……………………………………………………………………………120 材料與方法……………………………………………………………………122 結果……………………………………………………………………………129 討論……………………………………………………………………………139 結論…………………………………………………………………………………141 參考文獻……………………………………………………………………………14

Studies on Selective Maker Gene-Free Transplastomic Pak-choi (Brassica campestris L. ssp. chinensis (L.) Makino) for Transglutaminase Production

小白菜為台灣重要葉菜類，生長快速，對環境有很強的耐受性，因此對氣候土壤等自然環境相對限制小，栽培技術要求也相對低，在台灣各地全年皆可栽培。利用小白菜作為生物反應器，不僅可生產高經濟價值產品，提升其附加價值，更能利用其作物的生產特性，快速、簡易且較不受環境限制，大量生產目標蛋白，以供工業、食品、飼料及醫藥方面應用。 轉穀氨醯胺酵素 (Transglutaminase, TGase)催化蛋白質分子內與分子間ε-(γ-glutamyl) lysine共價鍵之生成，使蛋白質分子間形成架橋而成三次元結構，依其具蛋白濃縮液形成膠體化之能力，在食品加工上有很高的應用價值及潛力。以葉綠體作為基因轉殖的標的具有：增加轉殖基因大量表現，不造成基因污染、無基因靜寂及插入位置效應，較細胞核基因轉移穩定等優點。 本研究利用葉綠體DNA具有同源重組的特性，轉殖在同源重組DNA序列之間構築有轉榖氨醯胺酵素基因(transgluyaminase, tga)的目標基因，重組位置之外構築有丙氨酸消旋酵素基因(alanine racemase, AlaR)、D型胺基酸氧化酵素基因(D-amino oxidase, daao)、氨基葡萄糖苷腺苷轉移酵素基因(aadA)等篩選標誌基因的載體，到小白菜之葉綠體，預期經過二次DNA同源重組，則只會在葉綠體基因組內保留同源序列之間的tga目標基因，而剔除AlaR、daao、aadA等篩選標誌基因及殘存載體之DNA序列。本研究的目的為開發無篩選標誌基因且大量表現轉榖氨醯胺酵素之葉綠體轉殖小白菜的技術平台。 本研究已將自放線菌Streptomyces netropsis中篩選出的轉榖氨醯胺酵素基因(tga)，所構築到小白菜葉綠體基因轉殖之四種載體，pMT91EP-sRA、pMT91EP-sDA、pMT92GP-sDA及pMT92GP-sRA，利用基因槍法轉殖到''台農3號''小白菜之本葉的葉綠體。培殖體經100~200 ppm D-alanine或25~50 ppm spectinomycin交替漸進篩選及誘導再生，目前已獲得pMT91EP-sRA 及pMT92GP-sRA 轉殖之T0再生植株及其T1、T2子代。T0與T1葉片經PCR分析之結果顯示，tga基因已存在於轉殖之小白菜的葉綠體基因組中。T2葉片經PCR及RT-PCR分析之結果顯示，部份tga基因轉殖植株的AlaR及aada篩選標誌基因已被惕除，並可表現tga mRNA，且以西方墨點可偵測目標產物TGase之蛋白表現。本研究之結果顯示剔除篩選標誌基因之無篩選標誌基因的葉綠體基因轉殖技術，應用在小白菜葉綠體轉殖植株作為生物反應器，以生產轉穀氨醯胺酵素是可行的。Pak-Choi is one of the most important leafy vegetables in Taiwan. Pak choi is easy and fast to grow which makes it an ideal vegetable for summer season. It can be grown all year round in Taiwan. Utilization of Pak-choi as bioreactors not only can produce high economic product and increase the additional values, but also can take advantage of its cultural characteristic to produce large amount of target protein for industry, food, forage, and medical application. Transglutaminase (TGase) can catalyze the formation of ε-(γ-glutamyl) lysine covalent bond within molecules and between molecules and form 3D structure by cross link between proteins. On the basis of the gelatinize ability of protein concentrate, TGase has great application potential in food industry. Expression of target genes via chloroplast genomes not only enhances the level of expression without epigenetic effects, but also prevents out crossing of the introduced target genes via pollen grain. In this study, chloroplast transformation vectors harboring the selective marker gene (AlaR, daao, and aadA) and target genes (tga, gus, and egfp) constructed outside and of homologus recombination sequences, respectively, were transferred into the chloroplast of Pak-Choi via biolistic bombardment. After occurring twice of DNA recombination with chloroplast genome, only target gene will remain in the chloroplast genome and the selective gene and vector residues will be eliminated. The purpose of this study is to develop selective maker gene-free technique of Pak-choi chloroplast transformation for the production of TGase. In this study, 4 vectors pMT91EP-sRA, pMT91EP-sDA, pMT92GP-sDA, and pMT92GP-sRA, harboring the tga gene were transferred into the chloroplast of Pak-Choi ''Tainung No.3'' via biolistic bombardment. The regenerated plantlets were primary selected by 100~200 ppm D-alanine or 25~50 ppm spectinomycin. T0, T1, and T2 trasplastomic Pak-Choi plants were obtained from pMT91EP-sRA and pMT92GP-sRA constructs. The results of PCR, RT-PCR and western blots analyses indicated that the tga gene was integrated in the chloroplast genome of transplastomic Pak-Choi and expressed tga mRNA as well as TGase protein, while selective marker genes, AlaR, and aadA, were not detectable in the transplastomic Pak-Choi. Our results showed that the possibility of production of tga gene transplastomic Pak-Choi with maker gene-free technique.前言…………………………………………………………………………… 1 前人研究……………………………………………………………………… 4 一、葉綠體基因轉殖研究……………………………………………… 4 二、葉綠體基因轉殖應用………………………………………………5 三、篩選標示基因及剔除篩選標示基因在植物基因轉殖之研究進展 ………………………………………………………………………8 四、轉榖氨醯胺酵素之研究與應用……………………………………9 材料與方法……………………………………………………………………11 結果……………………………………………………………………………17 一、限制酵素切割分析轉殖載體………………………………………17 二、不同濃度之篩選藥劑對小白菜幼苗、子葉及本葉生育之影響 ……………………………………………………………………17 三、小白菜葉白菜植株之基因及表現分析…………………………19 討論…………………………………………………………………………… 56 一、不同濃度之篩選藥劑對小白菜幼苗、子葉及本葉生育之影響 ……………………………………………………………………56 二、小白菜葉綠體基因轉移之培殖體篩選、再生與後續子代繁殖…57 三、轉殖再生小白菜植株之基因及表現分析…………………………58 參考文獻……………………………………………………………………… 6

Using D-Alanine Racemase Gene (D-AlaR) as the Selectable Marker Gene for Cabbage(Brassica oleracea L. var. capitata L.) Chloroplast Transformation

甘藍 (Brassica oleracea L. var. capitata L.) 是世界重要經濟蔬菜，也是台灣栽培面積最廣的葉菜類蔬菜，有其重要的民生及經濟地位。過去五十年來，傳統育種工作對改良甘藍特質已有許多成效，但由於難以突破物種間基因資源的利用，使得在有限的種源親本間優良遺傳組合因子已達極限。發展甘藍基因轉殖技術，將可為改良甘藍品系提供一嶄新的途徑。植物葉綠體基因轉殖具有：增加轉殖基因大量表現，不會造成基因污染、基因靜寂及插入位置效應，與較細胞核基因轉移穩定等優點。因此開發葉綠體基因轉殖技術為近代生物技術的主力研發工作。由於葉綠體基因的大量表現轉殖基因，因此對非耐抗生素基因的篩選標誌基因的需求，更具必要性及急迫性。D型丙胺酸消旋酵素(D-alanine racemase, D-AlaR)藉由消旋化(racemization)反應，將D型丙胺酸催化形成L型丙胺酸。本研究以甘藍作為葉綠體基因轉殖研究之材料，探討以D-AlaR基因作為一安全性篩選標誌基因的可行性。並比較D-AlaR基因及aadA基因作為甘藍葉綠體基因轉殖篩選標誌之優劣。 本研究轉殖的載體攜帶有D-AlaR及aadA基因作為篩選標誌基因，egfp及gus作為報導基因，分別以prrn為啟動子，以psbA為終結子，命名為pMT91-RE(D-AlaR:egfp)、pMT91-REA (D-AlaR:egfp:aadA)、pMT91-RG (D-AlaR:gus)和pMT91-RGA(D-AlaR:gus:aadA)。利用基因槍法將其轟擊至''初秋''甘藍之下胚軸所誘導的癒傷組織，培殖體經4 mM D-alanine或15 ppm spectinomycin篩選6週後，平均存活率分別為27%及32%。但經spectinomycin篩選成活之培殖體無法再生成植株，最終死亡。D-alanine篩選成活之培殖體，獲得17株再生植株，其中14株具有D-AlaR基因。PCR及RT-PCR分析結果顯示，外源基因(D-AlaR、aada、gus、egfp)均已轉殖入甘藍葉綠體基因組中，並表現其mRNA。GFP綠色螢光及GUS活性染色分析結果，顯示在轉殖egfp或gus基因的植株中可觀察到綠色螢光的放射及藍色GUS反應。 綜合以上之結果顯示以D-AlaR基因作為甘藍之葉綠體基因轉殖的篩選標誌基因是可行的。以D-AlaR作為篩選標誌基因，配合D-alanine作為篩選藥劑，較aadA作為篩選標誌基因，配合spectinomycin抗生素作為篩選藥劑，具有容易判斷、篩選轉殖植株與少逃脫篩選的優點。Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetable crops grown worldwide, and also has been the most widely cultivated leafy vegetables in Taiwan. Traditional breeding methods have been very successful in making major improvements in the yield and quality of cabbage over the past fifty years; however, the traditional breeding methods have been limited by genetic resources. Currently, gene transformation offers new possibilities for improving the quality and quantity of cabbage. Chloroplast transgenic plants have several unique advantages, including increased the expression of transgene, not caused genetic pollution, affected silence gene and position of gene insertion, more stable gene transfer, etc. Therefore, the development of chloroplast transgenic techniques has become the main research and development in modern biotechnology. Because of the high expression of chloroplast transgene, the selection markers of non-antibiotic resistance gene have been urgently needed. D-alanine racemase (D-AlaR) catalyzes the racemization of D-alanine to produce L-alanine. This research is used cabbage as the material of chloroplast transgenic experiment, and D-AlaR gene as the feasibility of a safety selection marker gene. It also compares the fitness of D-AlaR gene and aadA gene as the selection marker of cabbage chloroplast transformation. This transformation research's vector carrying D-AlaR and aadA gene as gene selection markers, egfp and gus as reporter genes, Prrn as the promoter, and T-psbA as the terminator; named as pMT91-RE (D-AlaR-egfp), pMT91-REA (D-AlaR-egfp-aadA), pMT91-RG (D-AlaR-gus), and pMT91-RGA (D-AlaR-gus-aadA). The plastid transformation vector contained the gene clusters between the trnV--rrn16S and trnI--trnA--rrn23S regions, which was designed to be inserted into the chloroplast genome by homologous recombination after biolistic bombardment. We used biolistic bombardment to bombard the leaves or hypocotyls-induced calli of ''K-Y cross'' cabbage, and the survival rates of explants were 27% and 32% after six weeks of selection by 4 mM D-alanine or 15 ppm spectinomycin, respectively. There were no plants regenerated from survival explants which were selected by spectinomycin. Seventeen regenerated plants were obtained from the survival explants which were selected by D-alanine, fourteen of them contained the D-AlaR gene. The results of PCR and RT-PCR analysis indicated that transformed genes (D-AlaR, aada, gus, and egfp) were integrated into the plastid genome of transplastomic cabbage plants, and expressed its mRNA. GFP fluorescence and GUS histochemical staining analyses showed the emission of green fluorescence and blue-color reaction which were presented in the egfp or gus gene of transformed transplastomic cabbage plants. The results indicated that D-AlaR gene can be used as selection marker gene of cabbage's chloroplast transgene. D-AlaR as selection marker gene with D-alanine as selection agent is easier to determined transgenic plants and has fewer escaped-selection, compared to aadA as selection marker with spectinomycin as selection agent.中文摘要……………………………………………………………………… 1 英文摘要……………………………………………………………………… 2 前言…………………………………………………………………………… 3 前人研究……………………………………………………………………… 6 一、葉綠體基因轉殖之研究……………………………………………… 6 二、D型丙胺酸消旋酵素基因之研究……………………………………… 11 三、綠色螢光蛋白之研究…………………………………………………13 四、gus基因之研究…………………………………………………………17 五、aadA基因之研究………………………………………………………21 材料與方法………………………………………………………………24 結果……………………………………………………………………………31 一、轉殖載體分析…………………………………………………………31 二、D-alanine及Spectinomycin對未轉殖甘藍本葉及癒傷組織 再生之影響…………………………………………………………33 三、甘藍葉綠體基因轉殖之培殖體篩選及誘導再生………………………33 四、轉殖甘藍植株表現分析………………………………………………34 討論……………………………………………………………………………64 一、轉殖載體分析……………………………………………………………64 二、D-alanine及Spectinomycin對未轉殖甘藍本葉及癒傷組織 再生之影響……………………………………………………………65 三、甘藍葉綠體基因轉殖之培殖體篩選及誘導再生………………………65 四、轉殖甘藍植株之基因及表現分…………………………………………67 五、結論………………………………………………………………………69 參考文獻………………………………………………………………………7

Studies on Transformation of Functional Peptide VVYP Genes with Hypotriglyceridemic Action into Transplastomic Rice

Rice(Oryza sativa L.) is one of the most important crops grown worldwide, and also is the staple food and vital crop for the inhabitants in Taiwan. Currently, gene transformation offers new possibilities for obtaining high yielding, better quality rice cultivars, and also those resistant to major diseases. Using transgenic crops as bioreactors to produce industrial, feed and fodder additives, and pharmaceutical proteins become a new way for increasing economic values of crops and may solve the problems we are facing. Tetra-peptide, VVYP, is known to inhibit fat absorption in the digestive tract and to enhance the activity of hepatic triglyceride lipase to increase the clearance of body fat. Expression of foreign genes via chloroplast genomes offers several unique advantages, including high protein levels, expressing multiple genes in operons, transgene containment by maternal inheritance, as well as lack of gene silencing, position and pleiotropic effects. In this study, Gy5-9VVYP, βC-7VVYP and15VVYP genes are transformed into therice chloroplast by using daao/aadA gene as selectable marker gene. The objective of this study is to develop the rice chloroplast as a bioreactor for production of functional VVYP peptide with hypotriglyceridemic action. Chloroplast transformation vectors harboring the VVYP and daao/aadA genes had been constructed and transferred into the rice chloroplast via biolistic bombardment. The regenerated transplastomic plantlets were selected by D-alanine and spectinomycin. The results of PCR and RT-PCR analysis of T0 and T1 leaves indicated that the transformed genes are present in the chloroplast genome of transplastomic rice plants, and expressed its mRNA.水稻是世界重要經濟農作物，也是台灣最重要的糧食作物，有其重要的民生及經濟地位。過去五十年來，傳統育種工作對改良水稻品質與產量已有許多成效。應用生物科技轉移特定基因，可突破物種間雜交障礙，發展水稻基因轉殖技術，將可為改良水稻品系提供一嶄新新途徑。利用基因轉殖作物為生物反應器以生產工業、食品、飼料及醫藥方面的產品，在高附加價值、低成本及環保因素考量之下，是目前先進國家生物科技公司積極研究發展的重要項目。台灣土地面積及天然資源有限，因此極適合朝植物生物反應器的方向發展。因此本研究開發水稻做為生物反應器的系統，增加種植水稻的保健功效及附加經濟價值，以提昇台灣水稻產業的競爭力。 VVYP胜肽(Val-Val-Tyr-Pro) 能抑制消化道對於脂肪的吸收，以及增強肝臟中三酸甘油脂水解酵素的活性，因此能快速清理體內脂肪以達到降血脂的效果。植物葉綠體基因轉殖具有：增加轉殖基因大量表現，不會造成基因污染、基因靜寂及插入位置效應，與較細胞核基因轉移穩定等優點；因此開發葉綠體基因轉殖技術為近代生物技術的主力研發工作。 本研究計畫以daao及aadA (對照)基因作為篩選標誌基因，轉移經改造過含多套組VVYP之大豆儲藏性蛋白基因(Gy5-9VVYP、7Sb-7VVYP、15VVYP)至水稻葉綠體中。本研究之目的為探討利用水稻葉綠體為非耐抗生素篩選標誌基因的生物反應器，大量生產降血脂機能性胜肽VVYP的可行性，以提高水稻之保健功效及附加經濟價值。本研究已完成構築攜帶有多套VVYP胜肽為目標基因(Gy5-9VVYP、7Sb-7VVYP、15VVYP)，daao/aadA為篩選標誌基因之水稻葉綠體轉殖載體，並以基因槍法將所構築之基因轉殖到水稻葉綠體葉片。以D-alanine或spectinomycin篩選轉殖培殖體並誘導再生成水稻植株。轉殖再生植株T0及T1葉片之PCR及RT-PCR分析之結果顯示，轉殖之改造VVYP基因已存在於轉殖及之葉綠體基因組，並表現其mRNA。中文摘要…………………………………………………………………………………i 英文摘要………………………………………………………………………………ii 目次……………………………………………………………………………………iii 圖目次…………………………………………………………………………………vi 前言……………………………………………………………………………………1 前人研究………………………………………………………………………………3 一、活性胜肽－VVYP……………………………………………………………3 二、D型胺基酸氧化酵素……………………………………………………3 三、葉綠體基因轉殖………………………………………………………4 四、葉綠體作為生物反應器……………………………………………………6 材料與方法……………………………………………………………………………8 結果……………………………………………………………………………………15 一、 帶有改造9套VVYP－Gy5、7套VVYP－7Sb 大豆蛋白基因及15套VVYP基因為目標基因，daao或aadA為篩選標誌基因，之水稻葉綠體 轉殖載體之構築……………………………………………………………15 二、誘導水稻癒傷組織、基因槍轉殖、篩選及再生水稻植株………………20 三、轉殖再生水稻T0、T1植株之基因分析……………………………………20 討論……………………………………………………………………………………65 參考文獻………………………………………………………………………………6

Studies on Rice Chloroplast Transformation for Transglutaminase Production

Rice (Oryza sativa L.) is one of the most important and most widely cultivated crops inTaiwan. However, because the cost of cultivation is increasing every year, and the strongcompetition of imported rice from other countries after joining the WTO, the economic valueof growing rice is downsizing. In addition to reduce the production cost, use rice plants asbioreactors to produce industrial, feed and fodder additives, and pharmaceutical proteinsbecomes a new way for increasing economic values of rice and may solve the problems weare facing.Transglutaminase (TGA) catalyses an acyl-transfer reaction in which the γ-carboxamidegroups of peptide-bound glutaminyl residues are the acyl donors. The enzyme catalyses invitro cross-linking in whey proteins, soya proteins, wheat proteins, beef myosin, casein andcrude actomyosin refined from mechanically deboned poultry meat. In recent years, on thebasis of the enzyme's reaction to gelatinize various food proteins through the formation ofcross-links, this enzyme has been used in attempts to improve the functional properties offoods. Up to now, commercial TGA has been merely obtained from animal tissues. Thecomplicated separation and purification procedure results in an extremely high price for theenzyme, which hampers a wide application in food processing.Expression of foreign genes via chloroplast genomes not only dramatically enhances thelevel of expression and absence of epigenetic effects, but also prevents out cross of theintroduced foreign genes via pollen grains. Thus, transformation of the plastid genome is anew and attractive alternative to engineering the nuclear genome. Chloroplast transformationvectors contain a selective marker, most commonly a spectinomycin resistance (aadA) gene.Possible problems associated with plastid marker genes are the metabolic burden imposed byhigh-level expression and the potential for the unlikely event of horizontal transfer tomicrobes. D-amino acid racemase can mediate the racemization of D-amino acids. D-aminoacid oxidase catalyzes the dehydrogenation of D-amino acids to yield α-keto acid andaccompany with NH3 and H2O2 production. In this project, the possibilities of usingD-alanine racemase and D-amino acid oxidase genes as selection marker genes in ricechloroplast gene transformation are studied. The objective of this study is to develop therice chloroplast as a bioreactor with antibiotic-free selective maker gene to overproducetransglutaminase.水稻是台灣重要的農作物之一，其栽培面積十分廣大。但因栽培成本不斷提高，使得種植水稻之經濟效益相對降低。加上台灣加入世界貿易組織，市場開放，水稻的經濟價值面臨重大的壓力。因此，除了研究降低生產成本之外，提高水稻生產之附加價值，利用水稻當作生物反應器來生產高經濟價值生產工業、食品、飼料及醫藥方面的產品，成為另一個提升水稻價值之有效途徑。轉穀氨醯胺酵素 (Transglutaminase, TGA)會使蛋白濃縮液形成膠體化，因此在食品加工上有相當高的應用價值及潛力；例如：在漢堡、肉丸、魚漿、豆腐、植物蛋白粉末等可改善彈性、質地、口感、風味，並可增加儲存壽命。因此在食品加工上有相當高的應用價值及潛力，目前TGA 大多自動物肝臟中分離純化取得，價格相當高，一單位約八十美元，因此限制了其在食品加工上之應用植物葉綠體基因轉殖具有：增加轉殖基因大量表現，不會造成基因污染、基因靜寂及插入位置效應，與較細胞核基因轉移穩定等優點；因此開發葉綠體基因轉殖技術為近代生物技術的主力研發工作。由於葉綠體基因的大量表現轉殖之基因，因此對非抗抗生素基因的篩選標誌基因的需求，更具必要性及急迫性。D 型胺基酸氧化酵素(DAAO)可以催化D 型胺基酸的脫氨氧化反應(oxidative deamination)。D-型胺基酸消旋酵素可將D型組態的胺基酸轉換成L 型組態。本研究計畫擬以D-丙胺酸消旋酵素(D-AlaR)及D 型胺基酸氧化酵素(daao)基因作為篩選標誌基因，將轉穀氨醯胺酵素基因(tga)轉移至水稻葉綠體中。本研究之目的為探討利用水稻葉綠體為非抗抗生素篩選標誌基因的生物反應器，生產轉穀氨醯胺酵素的可行性，以提高水稻經濟效益

Development of marker-free transgenic plants with resistance to Tomato leaf curl Taiwan virus and Tomato spotted wilt virus

摘要 由粉蝨傳播的雙生病毒與由薊馬所傳播的番茄萎凋病毒是目前極為重要的兩群植物病毒。利用基因工程技術藉由基因轉殖的方式為目前有效的抗病毒的策略之一。然而在轉殖的過程中，通常都會使用抗生素抗性基因或殺草劑抗性基因作為篩選用的標誌基因以區分其中少數具有轉基因的植物。但是站在生態與食品安全的角度考量，對於使用此類抗性基因的安全疑慮越來越受大眾矚目，因此本論文的主旨即是建構可穩定移除標誌基因的轉殖系統，並利用此系統生產可抗雙生病毒與番茄萎凋病毒的無篩選標誌基因之轉基因植物。本研究所分析的雙生病毒是主要危害台灣番茄生長的台灣番茄捲葉病毒，首先，為了得知基因片段中何者可提供轉基因植物對雙生病毒表現較高的抗性，因此將台灣番茄捲葉病毒全基因體分割為數個基因片段後，再分別轉殖至圓葉菸草中進行抗性分析，發現其中四個構築可提供轉基因植物較高的抗性分別為：IRC1(基因間區域與C1開放讀碼區5′ 端之序列)，C2 (部分C2開放讀碼區之序列)，C2C3 (C2與C3開放讀碼區重疊區域之序列) 以及Rep2 (C1開放讀碼區之3′ 端序列)；並藉偵測轉基因植物中所累積的siRNA確認其抗病機制是經由基因沉寂所誘發。除此之外，番茄斑萎病毒是屬於Tospovirus屬而且擁有最廣泛寄主範圍的植物病毒之一，故此研究利用其N基因片段與台灣番茄捲葉病毒的部分C2開放讀碼區連結在一起作為一個嵌合轉基因，再將此構築轉殖入圓葉菸草與番茄中，以期能發展具有多重抗性之轉基因植物。經抗性分析後發現轉基因植物以農桿菌接種台灣番茄捲葉病毒後不會產生病徵，而機械接種番茄斑萎病毒後亦呈現高度抗性的現象，進一步分析抗病的轉基因植物則可偵測到siRNA的累積，證明此抗性是經由基因沉寂的機制所提供。同時也證實藉由連結不同基因體組成的病毒基因片段的策略可提供轉基因植物對DNA與RNA病毒具有抗性。與此同時，本論文亦以共轉殖法發展出三種生產無篩選標誌基因轉基因植物的轉殖系統，包括 (1) pGANP-CP1/pBin19：單一菌株內兩個獨立的質體各自攜帶有目標基因或標誌基因的T-DNA；(2) pGA2T-CP1：同一質體中同時帶有目標基因及標誌基因的T-DNA；(3) 為了發展可適用更多植物種類的載體，尤其是對kanamycin敏感度較低的作物，因此構築了pGA2TNH：單載體攜帶兩組T-DNA而於其中一組T-DNA中帶有兩種不同的選擇性標誌基因。經此三種系統再生之轉基因菸草的共轉型效率皆很相近，約為於50%。至於兩組T-DNA於子代的分離現象亦符合預期，於單一標誌基因重複數的轉基因植物中，移除標誌基因的比例在雙載體系統為24.1%，單載體系統可達17.5%~18.6%。結果顯示這些系統確實可行且同樣都能有效率地移除選擇性標誌基因，進一步可提供簡便及實用性兼具的工具應用至生產無篩選標誌基因的轉基因植物。因此，本論文將先前研究結果所提及之可提供轉基因植物對台灣番茄捲葉病毒產生較高抗性的基因片段，包括IRC1、C2、C2C3、Rep2並與番茄斑萎病毒之部分N基因片段連結在一起後，構築至二位元載體pGA2TNH藉此生產具有病毒抗性的無篩選標誌基因轉基因菸草。並利用農桿菌接種法篩選出對台灣番茄捲葉病毒具有抗性的轉基因親本植物，且經由自交的方式於其子代中獲得無篩選標誌基因轉基因抗病菸草。總而言之，本論文的結果提供了可應用在田間防治雙生病毒與番萎凋病毒的轉基因策略。而所研發的生產無選擇性標誌基因轉基因植物的轉殖系統，也將有助於提升一般大眾對轉基因作物的接受度。Abstract Whitefly-transmitted geminiviruses (Geminiviridae) and thrips-borne tospoviruses (Bunyaviridae) are two groups of extremely important plant viruses. Current transgenic approach based on genetic engineering technology provides an efficient strategy to breed plants to resist viral infection. However, public concerns about the use of antibiotic- and herbicide-resistance genes for the selection of transgenic plants during the transformation process have increased tremendously. Therefore, the objectives of this study were to develop the reliable transformation system that could remove selectable markers while generating transgenic plants that would resist the infection of geminiviruses and tospoviruses. Firstly, Tomato leaf curl Taiwan virus (ToLCTWV), a predominant tomato-infecting geminivirus in Taiwan, was subjected to investigate which viral gene fragments can confer high resistance to geminiviruses in transgenic plants. Individual transgenic constructs covering the entire ToLCTWV genome was transformed into Nicotiana benthamiana plants. Four constructs including IRC1 (intergenic region flanked with 5' end of C1), C2 (partial C2 ORF), C2C3 (overlapping region of C2 and C3 ORFs) and Rep2 (3' end of the C1 ORF) of high resistance for ToLCTWV have been observed. The detection of siRNA in transgenic plants confirmed that the mechanism of resistance was via gene silencing. Moreover, the middle half of the N gene of Tomato spotted wilt virus (TSWV), which is the type member of Tospovirus, was fused with the partial C2 ORF of ToLCTWV as the chimeric transgene and transformed into N. benthamiana and tomato to develop transgenic plants with multiple viral resistance. The transgenic plants remained symptomless post agro infected with ToLCTWV and exhibited high resistance to TSWV. The detectable siRNAs demonstrated that the resistance was mediated by gene silencing mechanism. The results also explained that linking multiple gene fragments of two viruses with different genomic organization was an effective strategy to engineer plants against both DNA and RNA viruses. Meanwhile, we developed three strategies of co-transformation to generate marker-free transgenic plants; they were (1) pGANP-CP1/pBin19, which comprises two individual plasmids carrying T-DNA of the target and marker genes separately; (2) pGA2T-CP1, which consists of one plasmid carrying two T-DNAs for the target and marker genes; and (3) pGA2TNH, which contains two T-DNAs in one plasmid in which one T-DNA carries the bi-selectable marker which can be used for more plant species especially those with low sensitivity to kanamycin. The co-transformation frequencies of the R0 transgenic N. benthamiana plants for both selection marker and target gene were similar. The co-transformation frequencies of three vector systems were about 50%. Segregation of transgene and selectable marker gene was revealed in the progeny of some co-transformed lines. The highest production ratio of marker-free transgenic plants was 24.1% in two plasmids system, followed by 18.6% in one plasmid system and 17.5% in bi-selectable marker system. We demonstrated that these strategies were feasible and efficient to eliminate the marker genes, and can provide a practical and simple tool for generating marker-free transgenic plants. Therefore, previously mentioned gene fragments that confer high resistance to ToLCTWV including IRC1, C2, C2C3 and Rep2 were linked together and fused with the middle half of the N gene from TSWV to make a chimeric transgene to be constructed into the binary vector, pGA2TNH, for the generation of viral resistance marker-free transgenic N. benthamiana. The transgenic R0 plants resistant to ToLCTWV were obtained and the marker-free resistant progeny plants were segregated by self-pollination. Overall, the results showed in this study have important implications for field deployment of transgenic strategies to control geminivirus and tospovirus. Moreover, the plant transformation systems that can generate marker-free plants would certainly boost the public acceptance of transgenic crops.目次/Contents 摘要……………............……………………………………………...….……. i Abstract……….……………..………….…………...….…………………..… iii 目次/Contents…………………..………………..………..…………..……… v 表目次/Contents of table…………………………..…………………………. ix 圖目次/Contents of figure…………….…………………...….…………….... xi Chapter 1. Introduction and literature review…………………………..….. 第一章、前言及前人研究 1 雙生病毒之特性與經濟重要性……………………………………………... 2 Begomovirus的分子特性………………………………………………….… 2 Begomovirus各基因功能…………………………………….…………… 3 病毒複製策略………………………………………………….…………… 4 媒介昆蟲之傳播途徑………………………………………….…………… 4 雙生病毒之防治策略………………………………………………………... 4 轉基因植物防治病毒之策略機制…………………………………...……… 5 雙生病毒與基因沉寂機制…………………………………………...……… 7 發展抗雙生病毒植物之轉基因策略………………………...……………… 8 蛋白媒介抗性－表現病毒蛋白……………………………………...…… 8 RNA媒介抗性－基因沉寂………………………………………..……… 10 DNA干擾………………………………………………………………..… 11 表現非病毒蛋白………………………………………………………...… 11 番茄斑萎病毒屬 (Tospovirus) ………………………………...…………… 12 轉基因植物篩選標誌基因的生物安全性………………………...………… 13 無篩選標誌基因轉基因植物之轉殖系統……………...…………………… 14 共轉型法………………………………………………...…………………… 14 參考文獻………………………………………………...…………………… 18 圖表…………………………………………………...……………………… 35 Chapter 2. Construction for the production of marker-free transgenic plants via Agrobacterium-mediated transformation…………... 第二章、以農桿菌轉殖法開發與應用無篩選標誌轉基因植物之轉殖載體 39 Abstract………………………………………………………….…………... 40 Introduction………..………………………………………………………… 41 Materials and methods…………………………………..………………….. 43 Vector construction………………………………………………………… 43 Tobacco transformation……………………………………………………. 44 Polymerase chain reaction (PCR) …………………………………………. 44 Southern blot analysis……………………………………………………… 45 Progeny segregation analysis………………………………………………. 45 Vector backbone detection in marker-free transgenic plants………………. 45 Results………………………………………………………………………... 47 Construction of co-transformation vectors………………………………… 47 Frequency of co-transformation and molecular analysis of R0 plants……... 47 Segregation of marker-free transgenic plants……………………………… 48 Vector backbone detection in marker-free transgenic plants………………. 48 Discussion……………………………………………………………………. 50 References………………………………………………………...………….. 52 Tables and Figures…………………...……………………………………… 56 Chapter 3. Construction of the binary vector with bi-selectable markers for generating marker-free transgenic plants…………………. 第三章、生產無篩選標誌轉基因植物的多功能轉殖載體之開發應用 62 Abstract……………………………………………………………………… 63 Introduction………..………………………………………………………… 64 Materials and methods……………….……………………………………... 65 Vector construction………………………………………………………… 65 Tobacco transformation……………………………………………………. 65 Polymerase chain reaction (PCR) …………………………………………. 65 Southern blot analysis……………………………………………………… 66 Progeny segregation analysis………………………………………………. 66 Results………………………………………………………………………... 67 Construction of bi-selectable markers binary vector…………………….… 67 Frequency of co-transformation and molecular analysis of R0 plants…….. 67 Segregation of marker-free transgenic plants……………………………… 68 Discussion……………………………………………………………………. 69 References………………………...………………………………………….. 71 Tables and Figures……………………...…………………………………… 73 Chapter 4. Evaluation of virus resistance conferred by the fragments covering entire genome of Tomato leaf curl Taiwan virus in transgenic plants ………………………………………………..... 第四章、台灣番茄捲葉病全基因體之各基因片段轉殖植物抗性分析 79 Abstract………………………………………………………………….…... 80 Introduction……..…………………………………………………………… 81 Materials and methods……….……………………………………………... 83 Plasmid constructions……………………………………………………… 83 Plant transformation……………………………………………………...… 83 Viral resistance assay of transgenic plants by agro-infection……………… 84 Small interfering RNA analysis by Northern blot…………………………. 84 Results………………………………………………………………………... 85 Development of transgenic N. benthamiana plants expressing viral gene fragments…………………………………………………………………... 85 The ToLCTWV resistance of transgenic plants……………………….…… 85 Accumulation of siRNA in transgenic plant lines…………………..……... 86 Discussion……………………………………………………………………. 88 References……...…………………………………………………………….. 92 Tables and Figures……………………...…………………………………… 98 Chapter 5. Development of marker-free transgenic plants resistance to Tomato leaf curl virus through a chimeric transgene derived from multiple gene fragments…………………………………... 第五章、藉連結多基因片段之策略研發抗番茄捲葉病毒之無篩選標誌轉基因植物 104 Abstract…………………………………………………………………..….. 105 Introduction……………..…………………………………………………… 106 Materials and methods……..……………………………………………….. 108 Construction of marker-free transformation vectors………………………. 108 Plant transformation……………………………………………………...… 108 Transgenic plants selection by Polymerase chain reaction………………… 109 Resistant analysis of transgenic plant with agro-infection………………… 109 Southern blot analysis and segregation of marker-free transgenic plants…. 110 Northern blot……………………………………………………………….. 110 Results………………………………………………………………………... 111 Construction and transgenic plants screening………………………….….. 111 Resistant analysis of transgenic plant with agro-infection………………… 111 Southern blot analysis and segregation of marker-free transgenic plants…. 112 Northern blot…………………………………….…………………………. 113 Discussion……………………………………………………………………. 114 References…………………...……………………………………………….. 117 Tables and Figures……..………………………………………………….… 120 Chapter 6. Resistance to a DNA and a RNA virus in transgenic plants by using a single chimeric transgene construct ………….……….. 第六章、具DNA 及RNA病毒多重抗性轉基因植物之研發 126 Abstract……………………………………………………………………… 127 Introduction………..………………………………………………………… 128 Materials and methods…………..………………………………………….. 130 Silencing constructs………………………………………………………... 130 Plant transformation……………………………………………………….. 130 Screening transgenic plants by PCR……………………………………….. 131 Analysis of resistance to ToLCTWV and TSWV in C2 gene transgenic plants………………………………………………………………………. 131 Reverse transcription-polymerase chain reaction (RT-PCR) ……………… 132 Small interfering RNA detection of transgenic plants…………………….. 133 Results……………………………………………………………………….. 134 Construction and plant transformation…………………………………….. 134 Resistance analysis of ToLCTWV on R0 transgenic plants………………... 134 Resistance analysis of TSWV and ToLCTWV on R1 transgenic plants…… 134 Accumulation of siRNA in transgenic plants……………………………… 135 Discussion……………………………………………………………………. 136 References………………………………………………...………………….. 139 Tables and Figures………...………………………………………………… 143 Chapter 7. Conclusions…………………………………….………………… 第七章、結論 15