D型胺基酸氧化酵素基因(daao)作為甘藍葉綠體基因轉殖的篩選標誌基因之研究

Abstract

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.