【作者】 丁玉梅； 杨正安； 周晓罡； 张绍松； 孙茂林；

【Author】 DING Yu-mei~1 YANG Zheng-an~2 Zhou Xiao-gan~1 ZHANG Shao-song~1 SUN Mao-lin~(1*) 1.Key Laboratory of Agricultural Biotechnology of Yunnan,Kunming 650223,China; 2.College of Horticulture and Landscape,Yunnan Agricultural University,Kunming 650201,China

【机构】 云南省农业生物技术重点实验室； 云南农业大学园林园艺学院；

【摘要】 构建了苏云金杆菌晶体毒蛋白基因 Cry1A6的烟草叶绿体表达载体 pCTBt,该载体以烟草叶绿体基因组 trnI-trnA 为同源重组片段,含有壮观霉素抗性筛选标记基因 aadA、质体特异性启动子 Prrn 和终止子 TpsbA．利用基因枪转化法,将 pCTBt 导入栽培烟草红花大金元叶绿体,经过4次继代筛选,获得9个抗壮观霉素的转化株系,转化频率为0．3～0．6株/枪,其中有5个株系 PCR 检测结果为阳性,初步确定 Cry1A6基因已整合到烟草叶绿体中．转基因烟草抗斜纹夜蛾试验表明,幼虫死亡率为10％～75％．转基因烟草植株具有较好的杀虫活性．更多还原

【Abstract】 Chloroplast transformation has become an attractive alternative technology in higher plants.Here,we constructed an expression vector pCTBt used in chloroplast transformation of tobacco,which contains Bacillus crystal toxin Cry1Ab gene as an functional gene,trnI-trnA gene fragment from tobacco plastome as homologous targeting sequences,and the selectable marker gene aadA under the control of a strong plastid promoter Prrn and terminator TpsbA.The vector pCTBt was then bombarded into leaf explants of tobacco cuhivar’Red Flower Dajinyuan’using PDS- 1000/He biolistic particle delivery system.A total of 9 transgenic lines resistance to spectinomycin were obtained out of 20 plates bombarded with pCTBt after 16 weeks selection culture.The transformation efficiency(about one recovering plant from 2 or 3 bombardments)was lower compared to that of Maliga’s reports(one plant per bombardments).The PCR analyses showed that 5 out of 9 hnes performed positive,thus confirmed that the Cry1Ab gene were integrated into the chloroplast genome of tobacco.The bioassays showed the transgenic tobacco lines had a considerable resistance to the larvae of Prodenia litura at the lethal rate of 10% to 75%.更多还原