将GNA基因导入莴苣(L.sativa)及其表达的研究

Preliminary Study of the Introduction and Expression of GNA Gene in Lettuce (L. sativa)

将含CaMV35S启动子的雪花莲凝集素基因置换Ti质粒载体pBI121中的GUS基因得到了pBIGNA质粒，将其导入农杆菌LBA4404，得到LBA4404（pBIGNA）菌株．通过该农杆菌介导转化莴苣子叶，筛选得到了17个具有卡那霉素抗性的转化子，其中3个转化子PCR鉴定的结果为阳性，用Western杂交检测了这3个转化子中的GNA含量，结果表明第2号转化子中的GNA表达量为可溶性蛋白总量的0．4％．由于GNA在0．1％浓度时就对包括蚜虫在内的刺吸式昆虫具有显著的毒杀作用，因此推测此转基因莴苣可能具有抗蚜虫能力．另用DotWestern杂交检测LBA4404（pBIGNA）中GNA的表达量，排除了农杆菌污染的可能．

The plasmid BIGNA was constructed by replacing the GUS gene in pBI121 with Galanthus nivalis agglutinin gene containing CaMV35S promoter, and it was then introduced into Agrobacterium tumerfaciens LBA4404. The cotyledons of lettuce were transformed by this Agrobacterium strain. Seventeen Kanamycin-resistant transformant were regenerated.The presence of GNA gene in three of the putative transformants were confirmed by PCR,and the expression of GNA gene of the three transformants were analyzed by Western blot.In the No. 2 transformant, the GNA protein was detected at the level of 0. 4% of the total soluble protein. As the GNA at the expression level of 0. 1% was shown to be poisonous to sap-sucking insects such as aphids, it is expected that this lettuce transformant should show resistance to aphids to some extends.