农杆菌介导BtCry1Ac、NTHK1双价基因转化烟草植株的研究

Tobacco Transformation with Bivalent Gene BtCry1Ac and NTHK1 Mediated by Agrobacterium

为探讨多基因植物转化载体p096899中外源基因BtCry1Ac和NTHK1在转基因植株中的表达情况,通过根癌农杆菌介导的叶盘转化法转化野生烟草,经卡那霉素筛选,初步获得了10株转基因烟草植株。经PCR鉴定,证明目的基因已被整合到烟草基因组中。利用荧光定量PCR和ELISA分析目的基因在转录水平和翻译水平的表达量,并对转基因烟草株系进行了抗虫试验和耐盐性试验。结果表明,目的基因在转录水平和翻译水平分别得到表达,但表达量存在差异,其中2号转基因株系毒蛋白表达量最高,其含量达可溶性总蛋白的0.028 5%;饲虫试验中,部分转基因烟草株系对斜纹夜蛾的毒杀作用明显高于未转化烟草,其中2号转基因株系的抗虫性最高,饲喂第6天斜纹夜蛾幼虫死亡率达到75.56%,与Bt毒蛋白表达量相一致;在盐胁迫下,转基因烟草的叶片生长状况均好于对照(非转基因)烟草的生长。证明BtCry1Ac基因和NTHK1基因的转入在一定程度上提高了烟草的抗虫性和耐盐能力。

To reveal the expression of exogenous genes BtCry1Ac and NTHK1 of plant transformation vector p096899 harboring multi-genes in transgenic plants,the wild tobacco was transformed by Agrobacterium-mediated leaf disc transformation method.Ten strains of transgenic tobacco were initially screened out by kanamycin.Then,identified by PCR,the target genes were integrated into tobacco genome.Expression of target genes in transcription and protein levels were analyzed by real-time PCR and ELISA,and the strains of transgenic tobacco were also tested for insect-resistance and salt-resistance.The results showed that the target genes expressed in the transcription and protein levels,respectively,and there existed an expression amount difference among the strains.The toxic protein amount of the 2th strain was the highest,which accounted for 0.028 5% of the leaf total soluble proteins.Result of feeding insect experiment illustrated that toxic effect exerted by some of transgenic tobacco strains on Spodoptera litura was obviously stronger than that of untransformed strains（CK）.The 2th strain was most insecttolerant,and the mortality rate of tested larvae reached 75.56% on the 6th day,which was consistent with ELISA test results.Under salt stress,the growth state of the transgenic tobacco leaves was better than CK.These results prove that introduction of BtCry1Ac and NTHK1 genes into tobacco could improve the insect-resistance and salt-resistance.