转单、双Bt基因741杨外源基因表达和抗虫性比较

【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。

Comparison of exogenous gene expression and insect-resistance ability of transgenic 741 poplars with single and double Bt genes

【Aim】 To study the combining effects of two or more insect-resistance genes in plants, as well as to screen and confirm double Bt transgenic 741 poplar lines with high resistance against coleopteran and lepidopteran insects. 【Methods】 Eight lines with three transgenes (Cry3Aa+Cry1Ac+API), one line with double transgenes (Cry1Ac+API) and three lines with single transgene (Cry3Aa) of transgenic 741 poplar were used as experimental materials. Comparative studies were conducted from three aspects: PCR detection of exogenous gene, the expressions of insecticidal proteins and the assessment of insect-resistance ability of plant. 【Results】 The expected electrophoretic bands from the transgenic lines appeared in PCR amplification. ELISA detection showed that the expression of insecticidal proteins was consistent with the exogenous genes in each line. Toxicity tests were performed in the laboratory with Plagiodera versicolora and Hyphantria cunea on fresh detached leaves. Transgenic poplar lines carrying different insect-resistance genes demonstrated selective resistance to target insects, but showed no toxic effects towards non-target insects. Transgenic 741 poplar lines with double Bt transgenes had double insect-resistance ability, and individual lines showed resistance ranging from high, medium to low. Five lines (pCCA1, pCCA2, pCCA5, pCCA6 and pCCA9) selected with high resistance against P. versicolora showed higher toxicity than three single Cry3Aa gene lines with high resistance (pCC11, pCC53, and pCC84). As regard to resistance toward H. cunea, seven lines (pCCA2-pCCA7 and pCCA9) exhibited similar effectiveness as the single Bt line (pB29) and only one line (pCCA1) showed an extremely low level of resistance. 【Conclusion】 The combination of multiple insect resistance genes in 741 poplars exerts gene stacking effects which not only expand its insect-resistance spectrum but also improve its insect-resistance ability largely.