【作者】 高建华；

【导师】 沈志成；

【作者基本信息】 浙江大学 ， 环境生物学， 2011， 博士

【摘要】 随着转Bt(苏云金芽孢杆菌,Bacillus thuringiensis)基因抗虫作物的广泛应用,人们对昆虫抗性发生的忧虑日益激增,对抗性管理的研究也越来越深入。目前在分子水平使用的抗性管理策略主要是基因堆叠,即将两种或更多作用位点不同,无交叉抗性的Bt基因同时在一种作物中表达。这种策略已被证明能够显著延缓抗性发生。同时,多基因的使用,还可以使转基因作物同时应对多种害虫,可谓一举两得。基因堆叠有两种实现方式,一种比较常见,是将多种基因独立的转录表达。而另一种是将两种基因拼接起来,通过一个启动子转录表达。般认为,两种Bt基因融合的效果等同于两种亲本蛋白的叠加。cry1Ab和cry9Aa基因,氨基酸序列相似度低于30%,理论上讲受体结合位点可能不同,因此不会出现交叉抗性。我们选取这两个cry基因,去除两者原蛋白结构域ⅢC端的编码基因,保留其经典3结构域(Ⅰ,Ⅱ和Ⅲ)的活性部分。然后将两个截断的基因拼接起来,形成融合基因,cry1Ab-cry9Aa。大肠杆菌(Escherichia coli)表达的融合蛋白Cry1Ab-Cry9Aa生物测定显示,该蛋白对粘虫(Mythimna separate)和亚洲玉米螟Ostrinia furnacalis)具有很好的杀虫活性,其LC50远低于其亲本蛋白Cry1Ab和Cry9Aa。对棉铃虫(Helicoverpa armigera)和斜纹夜蛾(Spodoptera litura)的生测结果显示,CrylAb和Cry9Aa蛋白对其没有明显的杀虫或抑制作用,但融合蛋白Cry1Ab-Cry9Aa对两种昆虫具有显著生长抑制作用。其中,喂养Cry1Ab-Cry9Aa蛋白(5μg/cm2)的棉铃虫平均体重只有空白对照的十分之一,而斜纹夜蛾约为5分之一。我们进一步将Cry9Aa的N端59个氨基酸去除,得到Cry9AaDel蛋白。同时将Cry1Ab和Cry9AaDel融合形成Cry1Ab-Cry9AaDel蛋白。生物测定显示,Cry9AaDel完全失去对粘虫和亚洲玉米螟的活性,但是,相应的融合蛋白Cry1Ab-Cry9AaDel却依然保持对两种昆虫的高效杀虫活性。另外,Cry9AaDel对棉铃虫和斜纹夜蛾也无任何活性,但融合蛋白Cry1Ab-Cry9AaDel也表现出同Cry1Ab-Cry9Aa蛋白相近的抑制作用。为了测定两种融合蛋白对四种害虫的生物活性是否由于其亲本蛋白之间的协同作用,我们同时将各自的两种亲本蛋白混合进行生物测定。结果显示,CrylAb和Cry9Aa或Cry9AaDel之间并无协同作用。两种融合蛋白对四种害虫的生物活性,也远远高于混合处理。该结果说明我们构建的两种融合蛋白Cry1Ab-Cry9Aa和Cry1Ab-Cry9AaDel获得了更高的生物活性,具有潜在应用价值。为了进一步确认两种融合蛋白在转基因领域的应用潜力,我们将两种融合基因分别转化水稻品种粳稻秀水110。分析阳性转化水稻中融合蛋白的表达情况,发现两种融合蛋白在水稻中均能较好表达。不同转化株系之间表达量差异较大(0.2-2μg每克新鲜叶片)。另外,在水稻里表达的融合蛋白,部分被水稻自身的蛋白酶消化,降解为较小的片段,该现象也已有其它相似的报道。对四种害虫,棉铃虫、斜纹夜蛾、二化螟(Chilo suppressalis)和稻纵卷叶螟(Cnaphalocrocis medinalis)等一龄幼虫进行生物测定显示,两种转基因水稻都获得了优秀的抗虫能力。进一步使用单拷贝转化株系的T1代植株测定二龄斜纹夜蛾幼虫,也表现出很好的抗性。综上所述,获得了两种Bt融合蛋白Cry1Ab-Cry9Aa和Cry1Ab-Cry9AaDel。对比其亲本蛋白,该融合蛋白对部分鳞翅目害虫表现出更高的杀虫活性。另外,转基因水稻分析表明,两种融合蛋白能够为水稻提供良好的抗虫性能。因此,两种融合蛋白具有在转基因抗虫作物中的应用潜力。更多还原

【Abstract】 The Bt (Bacillus thuringiensis) crops has been planted worldwide for control of insect pests. Recently, several Bt-resistant insect species has been discovered. For resistance management, many strategies have been reported. Gene stacking, i.e. expessing multiple toxins in a plant, can not only significantly delay the development of the insect resistance, but also confer resistance to multiple insect species to a plant. Constructing a translational fusion gene is an alternative way of using multiple toxins. And there were some researches focusing on evaluating different fusion Bt proteins expressed in either Escherichia coli or crops. At present, it was considered that the fusion Bt protein exert overlapping insecticidal activity and spectrum of the parental toxins.In this study, a fusion protein Cry1Ab-Cry9Aa deriving from the truncated CrylAb (648 amino acid residues at the N-terminus) and Cry9Aa (656 amino acid residues at the N-terminus), was expressed in E. coli. The two truncated toxins, CrylAb and Cry9Aa, include intact 3-domain structure and are sufficient for their insecticidal activities. The assay results showed that the fusion protein Cry1Ab-Cry9Aa is more active against the oriental armyworm (Mythimna separate) and the Asian corn borer (Ostrinia furnacalis) than the parental toxins, the truncated CrylAb and Cry9Aa. For cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura), the fusion protein significantly inhibited the growth of the neonate larvae and the feeding larvae in 5μg/cm2 concentration treatment were about 10% and 20% respectively in weight to the control insects. The larvae of cotton bollworm and tobacco cutworm feeding with truncated Cry1Ab or Cry9Aa almost developed normally. In addition, the assay results of the mixture treatment indicated that mixing the CrylAb and Cry9Aa cannot create any significant synergetic effect.The other fusion protein Cry1Ab-Cry9AaDel was made from the truncated Cry1Ab and Cry9AaDel, the further truncated Cry9Aa in which the 59 amino acids of the N-terminus was removed. Compared with the truncated Cry9Aa, the Cry9AaDel is inactive on the oriental armyworm and the Asian corn borer while it do not inhibit the growth of cotton bollworm and tobacco cutworm. However, the fusion protein Cry1Ab-Cry9AaDel retains similar insecticidal activity with the Cry1Ab-Cry9Aa to the four species tested. The two fusion genes were consequently introduced into a local cultivar of rice (Oryza sativa L. ssp. japonica), respectively. Western blot analysis revealed that the fusion proteins can be expressed reasonably in the transgenic rice in range from 0.2 to 2μg per gram of fresh leaves. And the fusion proteins expressed in rice were partially degraded, which was in agreement with the previous reports. These positive transgenic lines were highly resistant to four pest insects, cotton bollworm, tobacco bollworm, rice stem borer (Chilo suppressalis) and rice leaf folder (Cnaphalocrocis medinalis).In summary, the two fusion protein Cry1Ab-Cry9Aa and Cry1Ab-Cry9AaDel constructed in our study gain broader and higher insecticidal activity and can provide an excellent insect-resistance in biotech crops.更多还原