期刊
PLANT CELL TISSUE AND ORGAN CULTURE
卷 106, 期 1, 页码 63-70出版社
SPRINGER
DOI: 10.1007/s11240-010-9894-2
关键词
Taxol; Taxus chinensis cells; dbat; Transformation; Agrobacterium tumefaciens
资金
- National Natural Science Foundation of China [20776058]
- Ministry of Education of China
- National 11th Five-Year Plan to Support Science and Technology Project of China [2008BAI 63B04]
Taxus chinensis suspension cells were transformed by the Agrobacterium-mediated transformation method to overexpress the dbat gene coding for 10-deacetylbaccatin III-10 beta-O-acetyltransferase, a key enzyme for taxol biosynthesis. A. tumefaciens strain LBA4404 harboring either pCAMBIA1303 or the recombinant plasmid p1303-SdbatN was used. Both plasmids harbored the hygromycin phosphotransferase gene (hptII) and gusA-mgfp5 gene as selectable markers, but the latter plasmid also harbored the dbat gene in the T-DNA region. The transgenic T. chinensis cells had been maintained in modified Gamborg's B5 medium supplemented with hygromycin for more than 14 months and were subcultured at 4-week intervals. The selected transgenic cells were identified by PCR, Southern blot analysis, beta-glucuronidase assay and western blot analysis, and the results showed that the transgenes were integrated in the chromosomal DNA of T. chinensis cells with single-copy style, and that the gusA-mgfp5 reporter gene was expressed in the transgenic cells. The dbat mRNA expression level in the p1303-SdbatN-transgenic T. chinensis cells tested by real-time quantitative PCR was 5.3 +/- A 0.6 times that of the non-transformed cells. Taxol yield of the p1303-SdbatN-transgenic T. chinensis cells was about 1.7 times that of the non-transformed cells. These results suggest that the overexpression of dbat gene in transgenic T. chinensis cells leads to increased taxol yield.
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