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首页> 外文期刊>Journal of Biotechnology >Chloroplast-targeted expression of recombinant crystal-protein gene in cotton: An unconventional combat with resistant pests
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Chloroplast-targeted expression of recombinant crystal-protein gene in cotton: An unconventional combat with resistant pests

机译：棉花中重组晶体蛋白基因的叶绿体靶向表达：与抗性害虫的常规战斗

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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 T-3 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
机译：用单一Bt基因转化的植物很容易产生昆虫抗性，并且在全世界进行的大量Bt棉花商业化种植研究中已经报道了这一点。后来，为了转化抗性幼虫，设想转化具有多个Bt基因的植物。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，这可以延迟昆虫对特定Bt毒素的抵抗力的危害。但是，在田间条件下，很难在不经历重组作用的情况下，在许多世代后发展出均等表达所有Bt基因的纯合植物。在过去的十年中，许多声称声称能在单个植物中将植物从3个转基因转化为7个转基因的研究，但尚无一项申请纯合转基因品系的专利。更好的策略可能是使用经过修饰的杂交Bt基因，以改善凝集素结合结构域，以增强昆虫中肠中的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，并且在田间条件下开发纯合的转基因品系相对容易。杂交-Bt基因的叶绿体定位增强表达将使昆虫没有发展抗性的空间。我们设计并成功地将该策略应用于棉花（陆地棉），直到T-3代的数据表明我们的转基因棉花植物显示出针对叶绿体的Cry1Ac-RB表达增强。实验室和现场生物测定对美国棉铃虫（Heliothis armigera），粉红色棉铃虫（Pictinophora scutigera）和秋季夜蛾（Spodoptera frugiperda）均给出了令人鼓舞的结果，否则据报道它们对Cry1Ac毒素产生了抗性。通过ELISA确认从转基因棉花品系的叶中提取的富含叶绿体的蛋白质样品的水平升高了杂合Bt毒素的水平。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。

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