無篩選標誌基因之轉殖小白菜(Brassica campestris L. ssp. chinensis (L.) Makino) 葉綠體生產轉榖氨醯胺酵素之研究

Abstract

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 e-(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.