|摘要: ||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。
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.