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【目的】研究联合使用两种或两种以上的抗虫基因的抗虫效果,同时鉴定并筛选出转双Bt基因741杨对鳞翅目和鞘翅目害虫有较强抗性的株系。【方法】选取转三基因(Cry3Aa+Cry1Ac+API)741杨8个株系、转双基因(Cry1Ac+API)741杨1个株系和转单基因(Cry3Aa)741杨3个株系为试材,从外源基因PCR检测、毒蛋白表达和抗虫性三方面对转基因株系进行对比分析。【结果】经PCR扩增后各转基因株系出现了预期的电泳条带。ELISA蛋白检测显示转基因株系中都有与所含基因相应的Bt杀虫蛋白表达。用转基因株系新鲜叶片进行柳蓝叶甲Plagiodera versicolora和美国白蛾Hyphantria cunea室内饲虫实验表明:转入不同抗虫基因的杨树对昆虫的抗性具有选择性,对非靶标昆虫没有毒杀作用。转双Bt基因741杨具有双抗性,不同转基因株系表现出高中低的抗性水平:在对柳蓝叶甲的抗性上,筛选出的其中5个高抗株系(pCCA1,pCCA2,pCCA5,pCCA6和pCCA9)的抗性水平明显比含Cry3Aa单Bt基因的3个高抗株系(pCC11,pCC53和pCC84)高;在对美国白蛾的抗性上,有7个株系(pCCA2～pCCA7和pCCA9)的抗性水平与含Cry1Ac单Bt基因株系(pB29)表现一致,只有1个株系(pCCA1)对美国白蛾表现出了极低的抗性。【结论】多个抗虫基因在741杨上的联合使用,不仅扩大了抗虫谱,其中的高抗株系还具有了更高的抗虫能力,有效地发挥了基因的叠加效应。
【Objective】 The objective of this study was to investigate the combined effects of two or more insect-resistant genes and to identify and screen out transgenic Bt 741 poplar plants that are highly resistant to Lepidoptera and Coleoptera. 【Method】 Eight lines of transgenic calli (Cry3Aa + Cry1Ac + API) 741 were selected as experimental materials. Three lines of transgenic calli (Cry1Ac + API) 741 and three lines of transgenic calli (Cry3Aa) The transgenic lines were analyzed from three aspects of exogenous gene PCR, toxic protein expression and insect resistance. 【Result】 The expected bands of electrophoresis appeared in each transgenic line after PCR amplification. ELISA protein detection showed that the transgenic lines have the Bt insecticidal protein expression corresponding to the contained genes. The experiments of indoor feeding on Plagiodera versicolora and Hyphantria cunea using fresh leaves of transgenic lines showed that poplar transferred to different insect-resistant genes is selective for insects and non toxic to non-target insects effect. The transgenic Bt 741 poplar was double resistant and the transgenic lines showed high, medium and low resistance levels. Among the five resistant lines (pCCA1, pCCA2, pCCA5, pCCA6 and pCCA9) were significantly higher than the three highly resistant lines (pCC11, pCC53 and pCC84) containing the Cry3Aa single Bt gene; and 7 lines (pCCA2 ~ PCCA7 and pCCA9) were consistent with the Cry1Ac single Bt gene line (pB29), and only one line (pCCA1) showed extremely low resistance to H. armigera. 【Conclusion】 The combined use of multiple insect-resistant genes in 741 Yang not only broadened the spectrum of resistance to insects, but also increased the insect-resistant ability of the high resistance lines and effectively exerted the superposition effect of genes.