转双抗虫基因三倍体毛白杨外源基因表达及性状相关性分析

Expression of exogenous gene and correlation analysis of characters of transgenic triploid of Chinese white poplar carrying two insect resistance genes

用部分改造的BtCry I Ac基因与慈菇蛋白酶抑制剂（API）基因构建的双抗虫基因表达载体，通过农杆菌介导法转化了三倍体毛白杨Populus tomentosa Carr.，获得一批转双抗虫基因株系。对转基因株系的抗虫毒蛋白表达进行了ELISA和Westem Blot检测，同时用转基因株系叶片对杨扇舟蛾Clostera anochoreta Fabricius和舞毒蛾Lymantria dispar L．幼虫进行室内饲虫试验，并对各项外源基因表达指标进行了相关分析。结果表明：在检测的28个转基因株系中，对杨扇舟蛾高抗株系占总参试系号的41％，中抗系号占35．0％，低抗系号占24％，对舞毒蛾高抗株系占参试系号的70％。转基因植株可明显抑制存活幼虫的生长发育，且不同转基因株系饲养的幼虫发育存在显著差异。连续两年相关分析表明，不同转基因株系幼虫死亡率间存在极显著相关。转基因植株对舞毒蛾和杨扇舟蛾均表达出抗虫性，并存在极显著相关。ELISA检测结果表明，不同转基因株系Bt毒蛋白表达量存在差异，变化在0．0011％～0．0161％。转基因植株对害虫的杀虫效果与Bt杀虫蛋白的表达量存在显著相关，表明Bt毒蛋白在抗虫效果中占有重要地位。转基因植株对卡那霉素表现出一定的抗性，但与抗虫程度相关不明显，单纯用卡那霉素作为筛选手段并不能完全反映植株的真实抗虫效果。

Explants of the triploid of Chinese white poplar were transformed with Agrobacterium tumefaciens LBA4404 carrying the expression vector pBtiA containing two insect-resistant genes （ Bt Cryl Ac gene and API gene）. The expression of exogenous gene of transgenic subclones was analyzed by ELISA detection, Western blotting and laboratory bioassay with Clostera anachoreta Fabricius and Lymantria dispar L. The relationship among the characters of expression of exogenous gene was analyzed. The results showed that on average 41% of 28 transgenic subclones had a high insect resistance （larval mortality more than 80 % ） against C. anachoreta, 35 % of them had middle insect resistance （larval mortality 40 % - 80 % ） and 24 % of them had a lower insect resistance （larval mortality lower than 40%）. Also 70% of the transgenic subclones had a high insect resistance against L. dispar. Transgenic plant could obviously inhibit the growth and development of the larva, and the inhibition effect of the subclones was different. The expression of exogenous genes in transgenic plant was stable. Correlation among larval mortalities of the transgenic subclones was significant in the two years tested. The transgenic plant showed insect resistance against C. anachoreta and L. dispar, and the resulted larval mortalities in the two insects were significantly correlated. ELISA detection indicated that the expression level of Bt toxoprotein in different subclones was varied in 0.0011% -0.0161%. The obvious correlation between insect mortality and Bt toxoprotein content was proved. This suggested that Bt Cryl Ac gene plays an important role in insect resistance. The transgenic plant also showed some resistance against kanamycin. But the correlation between the resistances against insect and that against kanamycin was not significant. This suggested that selection only with kanamycin could not reflect the plant insect resistance exactly.