Zhou Y, Tian Y, Wu B, Mang K
Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Chin J Biotechnol. 1998;14(1):9-16.
The cDNAs of snowdrop lectin mature protein and its precursor protein, GNA12 and GNA34, were inserted downstream of a 35S promoter with a double enhancer and a "omega" fragment of TMV-RNA cDNA in the binary vector pBin438, or the phloem-specific CoYMV promoter in the vector pBcop1, respectively, resulting in the construction of four plant expression vectors pBGna12, pBGna34, pBCGna12, and pBCGna34. Leaf stripes of Nicotiana tabacum var. K326 were transformed with A. tumefaciens LBA4404 harboring the above expression vectors, respectively. PCR and Southern blot analysis showed that foreign GNA genes were inserted into the genome of transformed tobacco plants. Western blot analysis indicated that GNA could be expressed efficiently up to 0.08-0.15% of total soluble proteins in transgenic tobacco plants with pBGna34 or pBCGna34, while in those with pBGna12 and pBCGna12 GNA were hardly detected by immunoassay. The results from insect bioassay with a peach aphid (Myzus persicae) demonstrated that the transgenic plants of pBGna34 and pBCGna34 were aphid-resistant as shown by a 45-60% reduction in insect population density, with some individual transgenic lines being reduced by over 90%. In addition, it was evident that the 35S promoter with a double enhancer and CoYMV promoter had similar abilities to direct the GNA gene to express in transgenic tobacco plants, but because the CoYMV promoter drives the foreign gene in a phloem-specific expression manner, the transgenic plants of pBCGna34 showed higher aphid resistance.
将雪花莲凝集素成熟蛋白及其前体蛋白的cDNA，即GNA12和GNA34，分别插入二元载体pBin438中带有双增强子的35S启动子和TMV-RNA cDNA的“ω”片段下游，或载体pBcop1中的韧皮部特异性CoYMV启动子下游，从而构建了四个植物表达载体pBGna12、pBGna34、pBCGna12和pBCGna34。分别用携带上述表达载体的根癌农杆菌LBA4404转化烟草品种K326的叶条。PCR和Southern杂交分析表明，外源GNA基因已插入到转化烟草植株的基因组中。Western杂交分析表明，在含有pBGna34或pBCGna34的转基因烟草植株中，GNA能够高效表达，其表达量可达总可溶性蛋白的0.08 - 0.15%，而在含有pBGna12和pBCGna12的植株中，免疫检测几乎检测不到GNA。用桃蚜（Myzus persicae）进行的昆虫生物测定结果表明，pBGna34和pBCGna34的转基因植株具有抗蚜性，昆虫种群密度降低了45 - 60%，一些单个转基因株系的昆虫种群密度降低了90%以上。此外，很明显，带有双增强子的35S启动子和CoYMV启动子在指导GNA基因在转基因烟草植株中表达方面具有相似的能力，但由于CoYMV启动子以韧皮部特异性表达方式驱动外源基因，pBCGna34的转基因植株表现出更高的抗蚜性。
