作　　者： ; ; ; ; ;

机构地区： 河北农业大学

出　　处： 《林业科学》 2006年第9期61-68,共8页

摘　　要： 建立三倍体毛白杨叶片再生体系,用部分改造BtCry1Ac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三倍体毛白杨无性系,在含卡那霉素50 mg·L-1的分化培养基上诱导产生不定芽的叶片占18%,在生根培养基上对转基因植株做进一步筛选,获得50个转化再生株系.PCR检测表明：80%的植株呈现阳性反应,部分株系进行Southern Blot检测,证明外源基因已经插入杨树基因组中.用Bt毒蛋白抗血清进行ELSA检测,结果表明：7个转基因株系都有Bt杀虫蛋白表达,表达量最高的株系约占叶总可溶性蛋白的0.016 1%.用经分子生物学检测的28个转基因株系叶片进行舞毒蛾和杨扇舟蛾幼虫饲虫试验,结果表明：不同株系幼虫杀死率明显不同,有39.3%的株系对舞毒蛾和杨扇舟蛾幼虫的致死率在80%以上;25.0%的株系对两种害虫的幼虫死亡率均在60%～80%之间;另有35.7%的株系幼虫死亡率在50%以下,有些株系基本未表现出抗虫性.同时具高抗虫性的转基因株系能够明显抑制存活幼虫的生长和发育. The regeneration system of the hybrid triploid of Chinese White Poplar was established and the partly modified two insect-resistant genes [ BtCrylAc gene and arrowhead proteinase inhibitor （API） gene] were transformed to the hybrid poplar clone. After transformation, 18% leaves could be induced shoots in specific medium contained 50 mg·L^-1 kanamycin. The transgenic plants were selected further on the MS medium used for root induction added 50 mg·L^-1 Kanamycin and 50 transformed subclones were obtained. The transformed plants got by reaping selection were detected by PCR and 80% took on positive reaction. Some subclones were tested by southern blot and proved the exogenous gene has inserted in the poplar genome. Leaves of transformed plant were detected by ELISA using antiserum. The result indicated that the Bt toxoprotein was found in 7 transgenic subclones and the proportion of the Bt toxoprotein expressing to the total fusible protein was 0.001 1 ~ 0. 016 1% , markedly higher than the control （0.000 6% ）. One-year old leaves of 28 subelones transformed with Bt-API genes successfully tested by molecular biological method, and were used to feed the Lymantria dispar and the Clostera anachoreta. Result indicated that 39.3% of tested plants （11 subclones） took on high resistance and the larvae mortality was over 80%; 25 percent （7 subclones） possessed relatively lower resistance and the mortality was 60% ~ 80% ; the other 10 subclones took on very low resistance with the mortality of below 50% and some even did not have any resistance. The transgenic plants with high insectresistance could also obviously inhibit the development of the larvae.

关 键 词： 三倍体毛白杨 双抗虫基因 遗传转化 抗虫性

领　　域： [农业科学] [农业科学]