转双价抗虫基因Bt-CpTI提高四倍体菘蓝对小菜蛾抗性  

Enhanced resistance of transgenic tetraploid Isatis indigotica by Bt and CpTI to moths

作　　者：丁如贤[1] 肖莹[2] 王凯[1] 陈万生[2] 张汉明[1] 张磊[1] 

机构地区：[1]第二军医大学药学院,上海200433 [2]第二军医大学长征医院,上海200003

出　　处：《中草药》2009年第4期621-624,共4页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(39870919,300709210)

摘　　要：目的将外源苏云金芽孢杆菌杀虫蛋白基因CryIA(c)和豇豆胰蛋白酶抑制剂基因CpTI共同转入四倍体菘蓝以获得鳞翅目昆虫小菜蛾抗性。方法构建了以CaMV 35S为启动子,新霉素磷酸转移酶(Npt-Ⅱ)为选择标记,带有CryIA(c)基因和CpTI基因的植物表达载体pGBI121S4ABC并转入根癌农杆菌LBA4404。采用叶盘法遗传转化四倍体菘蓝。转基因再生植株经PCR和Southern杂交检测,并进行抗小菜蛾实验。结果T0代转化四倍体菘蓝的双基因阳性转化率达到16.67%。Southern杂交检测结果表明外源CryIA(c)基因和CpTI基因均已经随机整合到转基因菘蓝的基因组中。与野生对照相比,转基因四倍体菘蓝对小菜蛾显示出明显抗性。结论转双价抗虫基因Bt-CpTI是提高四倍体菘蓝对小菜蛾抗性的有效手段。Objective Two insect resistance genes Bacillus thuringiensis （Bt） crystal protein gene CryIA（c） and cowpea trypsin inhibitor gene CpTI were introduced into tetraploid lsatis indigotica to enhance the resistance to moths. Methods I. indigotica was transformed with a plasmid, pGBI121S4ABC, containing CryIA（c） Bt and CpTI and the selectable gene （Npt- Ⅱ） driven by the CaMV35S promoter via Agrobacterium tumefaciens LBA4404 mediated transformation. Then PCR and Southern blotting assay were conducted followed by moth bioassay test. Results Co-transgenic rate of the two genes in tetraploid I. indigotica was 16. 67%. The integration and expression of introduced genes in To regenerated transgenic plants were confirmed by Southern blotting assays. Insect bioassay test demonstrated transgenic lines had significant inhibition to moths, compared with wild type control. Conclusion Co-transformation of Bt and CpTI genes is an effective strategy to enhance the resistance to moths for tetraploid I. indigotica.

关 键 词：四倍体菘蓝 根癌农杆菌 抗虫 苏云金芽孢杆菌(Bt)杀虫蛋白基因CryIA(c) 豇豆胰蛋白酶抑制剂(CpTI) SOUTHERN杂交 

分 类 号：R282.12[医药卫生—中药学]