To avoid the long time required for conventional sexual crossing, transgenic tobacco (Nicotiana tabacum L.) plants harboring phbA gene (encoding 3-ketothiolase） were used as the target plant for the second transformation mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn LBA4404 containing pZCB which was constructed by linking phbB (encoding acetoacetyl-CoA reductase), phbC (encoding PHB synthase) and ctp sequence to pBIB-HYG under the control of CaMV 35S promoter. The hygromycin resistant transformants were morphologically normal and stable integration of phbB and phbC was confirmed by PCR and PCR-Southern. Moreover, RT-PCR-DNA hybridization analysis showed that 6.67% of the transformed tobacco plants could express both phbB and phbC at transcriptional level.
二次转化获得整合phbA、PhB、phbC 基因的转基因烟草
张景昱 叶梁 李丽 宋艳茹*
（中国科学院植物研究所，北京100093）
摘要： 有导肽序列的phbB（编码乙酰乙酰CoA还原酶）和phbC（编码PHB合酶）连入pBIB_HYG得到组成型表达载体pZCB, 用冻融法转入根癌土壤杆菌(Agrobacterium tumefaciens (Smith et Townsend) Conn)并由其介导转化已整合且表达phbA（编码3-硫裂解酶）基因并具有卡那霉素抗性的转基因烟草(Nicotiana tabacum L.)。通过二次转化可避开传统杂交育种，在5个月内获得整合PHB合成所需3个基因的转基因烟草。所获转基因植株表型正常, 经PCR、PCR-outhern、RT-CR-NA杂交检测确定有50株烟草稳定整合phbB、phbC基因，其中6.67%的植株可在转录水平表达双基因。
关键词： 聚羟基丁酸酯；二次转化；转基因烟草
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