National Centre of Excellence in Molecular Biology, 87 West Canal Bank Road, Thokar Niaz Baig, Lahore 53700, Pakistan.
J Biotechnol. 2013 Jul 10;166(3):88-96. doi: 10.1016/j.jbiotec.2013.04.011. Epub 2013 Apr 30.
Plants transformed with single Bt gene are liable to develop insect resistance and this has already been reported in a number of studies carried out around the world where Bt cotton was cultivated on commercial scale. Later, it was envisaged to transform plants with more than one Bt genes in order to combat with resistant larvae. This approach seems valid as various Bt genes possess different binding domains which could delay the likely hazards of insect resistance against a particular Bt toxin. But it is difficult under field conditions to develop homozygous plants expressing all Bt genes equally after many generations without undergoing recombination effects. A number of researches claiming to transform plants from three to seven transgenes in a single plant were reported during the last decade but none has yet applied for patent of homozygous transgenic lines. A better strategy might be to use hybrid-Bt gene(s) modified for improved lectin-binding domains to boost Bt receptor sites in insect midgut. These recombinant-Bt gene(s) would express different lectin domains in a single polypeptide and it is relatively easy to develop homozygous transgenic lines under field conditions. Enhanced chloroplast-localized expression of hybrid-Bt gene would leave no room for insects to develop resistance. We devised and successfully applied this strategy in cotton (Gossypium hirsutum) and data up to T3 generation showed that our transgenic cotton plants were displaying enhanced chloroplast-targeted Cry1Ac-RB expression. Laboratory and field bioassays gave promising results against American bollworm (Heliothis armigera), pink bollworm (Pictinophora scutigera) and fall armyworm (Spodoptera frugiperda) that otherwise, were reported to have evolved resistance against Cry1Ac toxin. Elevated levels of hybrid-Bt toxin were confirmed by ELISA of chloroplast-enriched protein samples extracted from leaves of transgenic cotton lines. While, localization of recombinant Cry1Ac-RB protein in chloroplast was established through confocal laser scanning microscopy.
用单个 Bt 基因转化的植物容易产生抗虫性，这在世界各地进行的许多研究中已经有报道，这些研究在商业规模上种植了 Bt 棉花。后来，人们设想用多个 Bt 基因来转化植物，以对抗具有抗性的幼虫。这种方法似乎是有效的，因为各种 Bt 基因具有不同的结合域，可以延迟针对特定 Bt 毒素的抗虫性的可能危害。但是，在田间条件下，经过许多代的重组效应，很难开发出表达所有 Bt 基因的纯合植物。在过去十年中，有许多声称在单个植物中转化 3 到 7 个转基因的研究报告，但没有一个申请了纯合转基因系的专利。一个更好的策略可能是使用经过修饰的杂交-Bt 基因，以提高凝集素结合域，从而增强昆虫中肠的 Bt 受体位点。这些重组-Bt 基因将在单个多肽中表达不同的凝集素结构域，并且在田间条件下相对容易开发出纯合转基因系。杂交-Bt 基因在叶绿体中的增强表达将使昆虫没有机会产生抗性。我们设计并成功地将这一策略应用于棉花（Gossypium hirsutum），并在 T3 代的数据中表明，我们的转基因棉花植物表现出增强的叶绿体靶向 Cry1Ac-RB 表达。实验室和田间生物测定对美洲棉铃虫（Heliothis armigera）、粉斑棉铃虫（Pictinophora scutigera）和秋粘虫（Spodoptera frugiperda）具有令人鼓舞的效果，而这些昆虫据称已经对 Cry1Ac 毒素产生了抗性。通过从转基因棉花系叶片中提取的叶绿体富集蛋白样品的 ELISA 证实了杂交-Bt 毒素的水平升高。同时，通过共聚焦激光扫描显微镜确定了重组 Cry1Ac-RB 蛋白在叶绿体中的定位。
