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|標題:||Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment||作者:||Liu, C.W.
|關鍵字:||aadA gene;bombardment;cabbage;chloroplast transformation;uidA gene;complete nucleotide-sequence;green fluorescent protein;plant-based;vaccine;plastid transformation;transgenic chloroplasts;tobacco;chloroplasts;confers resistance;selectable marker;gene organization;expression||Project:||Plant Cell Reports||期刊/報告no：:||Plant Cell Reports, Volume 26, Issue 10, Page(s) 1733-1744.||摘要:||
The objectives of this research were first to isolate plastid gene sequences from cabbage (Brassica oleracea L. var. capitata L.), and to establish the chloroplast transformation technology of Brassica. A universal transformation vector (pASCC201) for Brassica chloroplast was constructed with trnV-rrn16S (left) and trnI-trnA-rrn23S (right) of the IRA region as a recombination site for the transformed gene. In transforming plasmid pASCC201, a chimeric aadA gene was cloned between the rrn16S and rrn23S plastid gene borders. Expression of aadA confers resistance to spectinomycin and streptomycin antibiotics. The uidA gene was also inserted into the pASCC201 and transferred into the leaf cells of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by 200 mg/l spectinomycin and streptomycin. After antibiotic selection, the regeneration percentage of the two cabbage cultivars was about 2.7-3.3%. The results of PCR testing and Southern blot analysis confirmed that the uidA and aadA genes were present in the chloroplast genome via homologously recombined. Northern blot hybridizations, immunoblotting and GUS histochemical assays indicated that the uidA gene were stable integrated into the chloroplast genome. Foreign protein was accumulated at 3.2-5.2% of the total soluble protein in transgenic mature leaves. These results suggest that the expression of a variety of foreign genes in the chloroplast genome will be a powerful tool for use in future studies.
|Appears in Collections:||生物醫學研究所|
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