万多荣1, 殷 娴1, 陈晓博1, 姜 洋1, 张海燕2, 肖尊安1*
(1.北京师范大学生命科学学院，中国 北京 100875；2.甘肃亚盛集团博士后科研工作站北京分站，中国 北京 100101）
摘 要：通过RT-PCR从美洲商陆叶片中获得PAP N端氨基酸修饰的cDNA克隆PAP-sp1。构建携带PAP-sp1的植物转化载体pBPAP，利用农杆菌介导将PAP-sp1导入烟草品种K326叶片细胞，通过抗性筛选、组织化学和PCR鉴定获得了转基因烟草植株，按形成转基因不定芽的外植体数统计，烟草K326的转化率高达8.1%。攻毒实验表明，与对照植株相比，转基因烟草株系发病推迟，感病程度较低，开花结果提早。
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cDNA Cloning of Mutant PAP Changed in Signal Peptide Sequence and Acquisition of Tansgenic Nicotiana Tabacum with High Transformation Efficiency
WAN Duo-rong1, YIN Xian1,CHEN Xiao-bo1, JIANG Yang1, ZHANG Hai-yan2, CHEN Zheng-hua2 , XIAO Zun-an1★
(1. College of Life Sciences of Beijing Normal University, Beijing 100875, China; 2. Postdoctoral Scientific Research Station of Gansu Yasheng Industrial (Group) Co., Ltd., Beijing 100101，China)
Abstract: A cDNA clone of PAP-sp1 with changed nucleotides within the N-terminal signal petide sequence was obtained by RT-PCR method from leaves of Phytolacca americana. The plant binary vector was constructed by inserting PAP-sp1 cDNA into the vector pBI121, which was then introduced into leaf explants of the tobacco variety K326 by Agrobacterium tumefaciens mediated method. Histochemical assay and PCR were employed to confirm the transgenic plantlets regenerated on screening medium, and the transformation efficiency was 8.1% counted by the number of regenerating explants. TMV mechanical infection test of the transgenic plants revealed that, in comparison to the control plants, the transgenic plants exhibited viral resistance by retarded symptoms of systemic infection, less mottled leaves and earlier blooming.
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