D型丙胺酸消旋酵素基因(D-AlaR)作為甘藍之葉綠體基因轉殖的篩選標誌基因之研究

Abstract

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.