双价抗虫基因植物表达载体的构建及其在烟草中的表达

Construction of Plant Expression Vector Harboring Two Insect resistant Genes and Their Expression in Tobacco

利用人工合成及密码子优化后的雪花莲凝集素 （ＧＮＡ） 基因， 与人工合成的ＧＦＭ ｃｒｙＩＡ构建了双价基因中间载体。双价基因进一步亚克隆到植物表达载体ｐＢＩ１２１．１上， 获得了带有两个抗虫基因ｃｒｙＩＡ及ＧＮＡ基因的高效植物表达载体ｐＧＷ４ＢＡＩ， 在载体中两基因的５′端、３′端各加入与在植物中能增强基因转录和翻译有关的调控元件。通过根癌农杆菌介导叶盘法转化烟草Ｋ３２６， 获得２８株卡那霉素抗性植株， 随机抽取１７株经ＰＣＲ及进一步Ｓｏｕｔｈｅｒｎ 印迹检测，证实了双价抗虫基因在烟草基因组中的整合。转基因烟草杀棉铃虫试验表明４０％ 的转基因烟草植株对棉铃虫具有高抗性； 抑制蚜虫试验表明有效降低了蚜虫繁殖率， 转基因植株抑制蚜口密度为３０％ ～８５％ 不等， 并且获得了数株兼抗棉铃虫及蚜虫效果均良好的转基因烟草植株。

The fully artificially synthesized Bacillus thurigiensis insecticidal protein gene cryIA and the synthesized GNA, (the mannose specific lectin from snowdrop (Galanthus nivalis)) were inserted into plasmid pBI121.1. The plant expression vector pGW4BAI was obtained, in which each gene in the vector was under the control of several regulation elements in relation to initiation and termination of transcription and enhancement of translation. Leave stripes of Nicotiana tabacum var. K326 were transformed with Agrobacterium tumaficiens strain LBA4404 harboring the plant expression vector. PCR and Southern blotting analysis showed that the foreign cryIA and GNA gene were inserted into the genome of transformed tobacco plants. Leaf disc bioassays against cotton bollworm (H. armigera) showed that transgenic tobacco plants had a high insecticidal activity. The inhibition of aphid population in leaf disc bioassays against Myzus prsicae showed that the fecundity of transgenic plants were lower than that on untransformed plants; the aphid population on the transgenic tobacco plants was 25%～70% of that on untransformed tobacco plant. Throuhg the two bioassays against H. armigera and M. prsicae, several transgenic tobacco plants showing high insect resistant activities to both pests were obtained.