基于Gene-Deletor系统的安全复合性状转基因烟草研究

The study of multi-genes in transgenic tobacco plants based on Gene-deletor system

本研究利用农杆菌介导法将带有水稻花粉特异启动子籼稻花粉过敏原基因(OSIPA)启动子驱动的Gene-Deletor外源基因清除系统,水稻Os GA3ox2(D18)启动子驱动水稻Os GA2ox1基因,玉米Ubiquitin启动子驱动BAR::GUS融合基因为筛选标记基因以及水稻Actin1启动子驱动驱动抗虫Cry1Ab基因复合性状植物表达载体p GM626-D18-Os GA2ox1遗传转化三星烟草。通过GUS组织化学染色及PCR分子鉴定,获得了43株转基因烟草植株。结果表明,水稻OsGA2ox1基因在烟草中表达能降低转基因烟草植株高度,但不影响植株生殖生长。转基因烟草对烟草斜纹夜蛾幼虫具有抗性,可抑制烟草斜纹夜蛾幼虫的取食与发育。以50 mg/L的除草剂Basta处理野生型和转基因烟草离体叶片,发现BAR基因提高了烟草抗除草剂的能力。对12株转基因烟草T0代花粉外源基因清除效率进行研究,发现部分烟草株系外源基因清除可达到100%,其他未完全清除外源基因株系的清除效率介于42.84%~99.97%之间。

In this study,the multi-gene expression vector pGM626-D18-OsGA2ox1 plasmid,which contained the rice pollen-specific expression promoter OSIPA of the Gene-deletor system,fusion selection marker gene BAR ： ：GUS under the control of maize Ubiquitin promotor,the Os GA2ox1 under the control of rice OsGA3ox2（ D18） promoter,and the Cry1Ab gene under the control of rice Actin promoter was transformed into the Xanthi tobacco using Agrobacterium tumefaction mediated method. Forty-three individual T0 transgenic tobacco plants were identified by GUS histochemical staining and PCR detection. The results showed that the expression of OsGA2ox1 gene in tobacco reduced the height of transgenic plants,but did not affect their reproductive growth. Larvae of Spodoptera litura（ F.） fed on transgenic plants showed reduced food ingestion and reduced growth and weight. The concentration of 50 mg/L basta was used for the herbicide sensitivity treatment. The expression of BAR gene enhanced the herbicide resistance in transgenic tobacco. The pollens from 12 individual T0 transgenic strains were used for the determination of excision efficiency test. The results showed that part of the tobacco strains with 100% excision efficiency,and the excision efficiencies in other lines were between 42.84 and 99.97%.