Xie Long-Xu, Xu Pei-Lin, Nie Yan-Fang, Tian Ying-Chuan
The Key Laboratory of Gene Engineering of Ministry of Education, Zhongshan University, Guangzhou 510275, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Sep;19(5):545-50.
A binary plant expression vector, pCM12-slm, carrying the aroAM12 mutant gene encoding bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and the Bts1m recombinant gene consisting of 331 N-terminal amino acids of CryIAc and 284 C-terminal amino acids of CryIAb has been constructed. The truncated Bts1 gene was fused with the PR1b signal peptide sequence and expressed in tobacco plants under the control of 2E-CaMV35S promoter and the omega (omega) translation enhancer sequence from tobacco mosaic virus. The mutant aroAM12 was fused with the transit sequence of tobacco EPSPS and expressed in tobacco plants under the control of the CaMV35S promoter. Tobacco leaves were transformed with Agrobacterium tumefaciens LBA4404 harboring the pCM12-slm plasmid, and the transgenic plants were selected directly on medium containing the herbicide. Forty glyphosate resistant plants were regenerated, with a transformation frequency of 27%. Transgenic plants were initially assessed for glyphosate resistance by placing leaf discs on shoot induction media containing the herbicide. Rooted plantlets, propagated from selected transgenic tobacco, were transferred to soil in a greenhouse and tested for glyphosate resistance by spraying them with Roundup at a commercial recommended dose. The glyphosate resistance assay indicated that all the transgenic plants showed highly resistant to the herbicide. The PCR assay showed that the aroAM12 gene was present in all of the 40 T0 transfer plants, and Bts1m genes present in 28 of 40 of the transgenic plants. Southern blot analysis further confirmed that the copy number of the transgenes varied from one to three copies in different transgenic plants. Northern blot and immunodot blot showed that the aroAM12 and Bts1m genes were expressed at the transcription and translation levels. Transgenic plants containing both the aroA M12 and Bts1m genes were further assessed for insect resistance. Tobacco leaves of T0 transgenic plants were infested with tobacco bollworm H. assulta larvae for 6 days. The result (table 1) showed that the survival rate of insect larvae was between 0-10%, and the growth of insect larvae was seriously inhibited, suggesting pCM12-slm as a dual functional vector with potential application in breeding of glyphosate and insect resistance transgenic plants.
构建了一个二元植物表达载体pCM12 - slm，其携带编码细菌5 - 烯醇丙酮酸莽草酸 - 3 - 磷酸合酶（EPSPS）的aroAM12突变基因以及由CryIAc的331个N端氨基酸和CryIAb的284个C端氨基酸组成的Bts1m重组基因。截短的Bts1基因与PR1b信号肽序列融合，并在2E - CaMV35S启动子和来自烟草花叶病毒的ω（omega）翻译增强子序列的控制下在烟草植株中表达。突变体aroAM12与烟草EPSPS的转运序列融合，并在CaMV35S启动子的控制下在烟草植株中表达。用携带pCM12 - slm质粒的根癌农杆菌LBA4404转化烟草叶片，并在含有除草剂的培养基上直接筛选转基因植株。再生出40株抗草甘膦植株，转化频率为27%。通过将叶盘置于含有除草剂的芽诱导培养基上来初步评估转基因植株对草甘膦的抗性。从筛选出的转基因烟草中繁殖出的生根小植株被转移到温室土壤中，并以商业推荐剂量喷洒农达来测试其对草甘膦的抗性。草甘膦抗性测定表明所有转基因植株对该除草剂均表现出高度抗性。PCR检测表明aroAM12基因存在于所有40株T0代转化植株中，Bts1m基因存在于40株转基因植株中的28株中。Southern杂交分析进一步证实不同转基因植株中转基因的拷贝数在1至3个拷贝之间变化。Northern杂交和免疫斑点杂交表明aroAM12和Bts1m基因在转录和翻译水平上均有表达。对同时含有aroA M12和Bts1m基因的转基因植株进一步评估其抗虫性。用烟草棉铃虫幼虫侵染T0代转基因植株的烟草叶片6天。结果（表1）表明昆虫幼虫的存活率在0 - 10%之间，且幼虫生长受到严重抑制，表明pCM12 - slm作为一种双功能载体在培育抗草甘膦和抗虫转基因植物方面具有潜在应用价值。
