D型胺基酸氧化酵素基因(daao)作為甘藍之農桿菌基因轉殖法的篩選標誌基因之研究

Abstract

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.