PDF(Pubmed)
Abstract：
BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants.
RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves.
CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.
RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves.
CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.
摘要：
背景：作为一项公共资助的计划的一部分，该计划旨在开发基因工程的芸苔（白菜，花椰菜,和油菜）为印度和澳大利亚农民表达苏云金芽孢杆菌晶体（Cry）编码的杀虫（Bt）毒素，我们设计了几个构建体,其驱动修饰的Cry1B和Cry1C基因(称为Cry1BM和Cry1CM;其中M表示修饰的)的高水平表达。修改这些基因的DNA序列的两个主要动机是将与表达CryM基因的转基因作物植物的商业种植相关的任何许可成本降至最低。并去除或改变可能对其在植物中的活性产生不利影响的序列。
结果：为了评估Cry1BM/Cry1CM基因的杀虫功效，将构建体引入拟南芥模型中，其中Cry1BM/Cry1CM表达从单（S4/S7）或双（S4S4/S7S7）地下三叶草特技病毒（SCSV）启动子指导。所得的转基因植物显示出高水平的Cry1BM/Cry1CM表达。Cry1CM的蛋白质积累范围为5.18至176.88µgCry1CM/g叶片干重。与以前关于特技促销员的工作相反，我们发现使用单或双特技启动子与Cry1BM/Cry1CM基因的表达水平之间没有相关性，从两个构建体中观察到相似范围的Cry1CM转录物丰度和蛋白质含量。以表达Cry1BM/Cry1CM基因的转基因拟南芥叶片为食的第一龄小菜蛾（小菜蛾）幼虫显示出100％的死亡率，转基因叶片的平均叶片损伤评分为0至5，其中转基因叶片为0.125，野生型叶片为4.2。
结论：我们的工作表明，修饰的Cry1基因适用于抗虫转基因作物的开发。除了美国的PAT基因，我们对这里描述的结构中组件的知识产权格局的评估表明，它们可以在不需要进一步许可的情况下使用。这能够显著降低将来在转基因作物植物中开发和使用这些Cry1M基因的成本。
结果：为了评估Cry1BM/Cry1CM基因的杀虫功效，将构建体引入拟南芥模型中，其中Cry1BM/Cry1CM表达从单（S4/S7）或双（S4S4/S7S7）地下三叶草特技病毒（SCSV）启动子指导。所得的转基因植物显示出高水平的Cry1BM/Cry1CM表达。Cry1CM的蛋白质积累范围为5.18至176.88µgCry1CM/g叶片干重。与以前关于特技促销员的工作相反，我们发现使用单或双特技启动子与Cry1BM/Cry1CM基因的表达水平之间没有相关性，从两个构建体中观察到相似范围的Cry1CM转录物丰度和蛋白质含量。以表达Cry1BM/Cry1CM基因的转基因拟南芥叶片为食的第一龄小菜蛾（小菜蛾）幼虫显示出100％的死亡率，转基因叶片的平均叶片损伤评分为0至5，其中转基因叶片为0.125，野生型叶片为4.2。
结论：我们的工作表明，修饰的Cry1基因适用于抗虫转基因作物的开发。除了美国的PAT基因，我们对这里描述的结构中组件的知识产权格局的评估表明，它们可以在不需要进一步许可的情况下使用。这能够显著降低将来在转基因作物植物中开发和使用这些Cry1M基因的成本。
