Liu Cheng-Wei, Lin Chin-Chung, Chen Jeremy J W, Tseng Menq-Jiau
Department of Post-Modern Agriculture, Ming Dao University, Chang Hua 523, Taiwan, ROC.
Plant Cell Rep. 2007 Oct;26(10):1733-44. doi: 10.1007/s00299-007-0374-z. Epub 2007 Jun 14.
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 IR(_A) 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.
本研究的目的首先是从甘蓝（Brassica oleracea L. var. capitata L.）中分离质体基因序列，并建立甘蓝的叶绿体转化技术。构建了一种用于甘蓝叶绿体的通用转化载体（pASCC201），以IR(_A)区域的trnV-rrn16S（左侧）和trnI-trnA-rrn23S（右侧）作为转化基因的重组位点。在转化质粒pASCC201中，一个嵌合的aadA基因克隆在rrn16S和rrn23S质体基因边界之间。aadA的表达赋予对壮观霉素和链霉素抗生素的抗性。uidA基因也被插入到pASCC201中，并通过粒子枪介导的转化转入甘蓝叶细胞。通过200mg/l壮观霉素和链霉素筛选再生苗。经过抗生素筛选，两个甘蓝品种的再生率约为2.7-3.3%。PCR检测和Southern杂交分析结果证实，uidA和aadA基因通过同源重组存在于叶绿体基因组中。Northern杂交、免疫印迹和GUS组织化学分析表明，uidA基因稳定整合到叶绿体基因组中。转基因成熟叶片中外源蛋白积累量占总可溶性蛋白的3.2-5.2%。这些结果表明，在叶绿体基因组中表达多种外源基因将成为未来研究的有力工具。
