转cry1Ab/cry2Aj和G10evo-epsps基因玉米中Bt蛋白的时空表达及抗性评价

【目的】探讨转cry1Ab/cry2Aj和G10evo-epsps基因玉米双抗505-12-5中外源Bt蛋白时空表达规律,对3种主要鳞翅目害虫亚洲玉米螟、黏虫和棉铃虫的抗性进行鉴定,为转基因玉米双抗505-12-5的商业化推广提供科学的数据支撑。【方法】Bt蛋白时空表达规律采用酶联免疫法(ELISA),田间抗虫性和室内抗虫性鉴定分别采用田间人工接虫和离体组织生测方法。【结果】在玉米6~8叶期,Bt含量表现为根心叶茎,分别为517.3、453.8和312.8 ng·g~(-1);大喇叭口期,Bt含量表现为心叶根茎,分别为353.3、281.3和232.9 ng·g~(-1);吐丝期,Bt含量表现为心叶根茎,分别为188.9、114.1和53.6 ng·g~(-1);乳熟期,根、茎和心叶含量相当,分别为178.0、160.3和185.4 ng·g~(-1);繁殖器官中Bt蛋白含量表现为籽粒花丝花粉雄穗,分别为181.3、100.1、95.0和79.8 ng·g~(-1)。室内抗虫性鉴定表明,转基因玉米双抗505-12-5心叶饲喂黏虫24 h,幼虫死亡率低,但48 h后达98.21%;双抗505-12-5心叶、花丝和籽粒饲喂玉米螟,24 h幼虫死亡率分别为87.37%、100%、100%;双抗505-12-5花丝饲喂棉铃虫,24 h幼虫死亡率达80.18%,48 h死亡率为92.45%。田间鉴定结果显示,转基因玉米双抗505-12-5在心叶期和雌穗期对玉米螟、心叶期对黏虫、雌穗期对棉铃虫的抗性均达高抗水平。【结论】转基因玉米双抗505-12-5各器官在不同生育期中均能表达Bt蛋白,尤其在鳞翅目害虫为害的主要时期6~8叶期和吐丝期及乳熟期,易受害器官中Bt蛋白表达量较高。转基因玉米双抗505-12-5田间及室内对3种鳞翅目害虫均表现了显著的抗性效果,具有推广应用的潜力。

Bt protein spatial-temporal expression and evaluation for resistance of transgenic cry1Ab/cry2Aj and G10evo-epsps maize

Aim] Bt protein spatial-temporal expression and insect-resistance to three Lepidoptera pests, Ostrinia furnacalis, Mythimna separata, and Helicoverpa armigera, were studied in order to provide scientific data for the commercialization of transgenic cry1Ab/cry2Aj and G10ev-epsps maize double resistant 505-12-5.Method] Spatial-temporal expression patterns of Bt protein were studied by ELISA. Field and laboratory insect resistance was tested by artificial grafting and in vitro tissue bioassay.Result] At different developmental stages, the content of Bt proteins significantly varied in the different organs. At 6-8 leaf stage, Bt protein contents were:root (517.3 ng·g^-1) > leaf (453.8 ng·g^-1) > stem (312.8 ng·g^-1). At the bell stage, Bt protein contents were:leaf (353.3 ng·g^-1) > root (281.3 ng·g^-1) > stem (232.9 ng·g^-1). At the silking stage, Bt protein contents were:leaf (188.9 ng·g^-1) > root (114.1 ng·g^-1) > stem (53.6 ng·g^-1). At the milky stage, Bt protein contents were similar in three organs, root (178.0 ng·g^-1), stem (160.3 ng·g^-1) and leaf (185.4 ng·g^-1). Bt protein contents in reproductive organs were:seed (181.3 ng·g^-1)> filament (100.1 ng·g^-1)> pollen (95.0 ng·g^-1)> tassel (79.8 ng·g^-1). The results of laboratory insect-resistant test showed that armyworm mortality rates were low after 24 h feeding with young leaves of 505-12-5, and reached to 98.21% mortality after 48 h of feeding. The corn borer larval mortality rates were 87.37%, 100% and 100%, respectively, after 24 h feeding with young leaves, filaments, and seeds of 505-12-5. The mortality rate of cotton bollworm was 80.18% after 24 h of feeding with filaments of 505-12-5 and 92.45% after 48 h. Insect resistance tests in the field also showed that 505-12-5 was highly resistant to corn borer in whorl stage and female ear stage, to armyworm in whorl stage, and to cotton bollworm at silking stage.Conclusion] Bt protein expressed in different organs of transgenic maize double resistance 505-12-5 at different growth stages. The expression of Bt protein was higher in pest susceptible organs at 6-8 leaf, silking and milky stages. Double resistant 505-12-5 transgenic maize has the potential for application because of its significantly high resistance to three Lepidoptera pests in the field and laboratory.