Gayen Srimonta, Mandal Chandi Charan, Samanta Milan Kumar, Dey Avishek, Sen Soumitra Kumar
Department of Human Genetics, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Biochemistry, School of Life Sciences, Central University of Rajasthan, Ajmer, 305817, India.
World J Microbiol Biotechnol. 2016 Apr;32(4):62. doi: 10.1007/s11274-016-2013-8. Epub 2016 Feb 29.
Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61 % mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW.
抗性昆虫的出现限制了苏云金芽孢杆菌（Bt）转基因作物在虫害治理方面的可持续性。除此之外，转基因作物中存在不需要的标记基因也是一个重大的环境和健康问题。因此，培育表达具有不同致死机制的新型毒素的无标记转基因作物对于抗性治理是必要的。在之前的一项研究中，我们构建了一种工程化的Cry2Aa（D42/K63F/K64P）毒素，与第一代Bt毒素Cry1A相比，它具有不同的致死机制，并且这种工程化毒素对作物的主要鳞翅目害虫的毒性提高了4.1至6.6倍。在本研究中，我们测试了这种工程化合成Cry2Aa（D42/K63F/K64P）毒素作为抗虫转基因烟草植物开发候选物的效力。同时，我们利用Cre/lox介导的重组方法从表达Cry2Aa（D42/K63F/K64P）的烟草植物中消除了选择标记基因，并成功培育出表达工程化Cry2Aa毒素的无标记T2转基因烟草植物。实时和蛋白质免疫印迹分析证明了转基因植物中工程化毒素基因的表达。昆虫取食试验表明，表达Cry2Aa（D42/K63F/K64P）毒素的转基因植物的无标记T2后代对棉铃虫（CLW）和棉铃象鼻虫（CBW）的死亡率分别为82%至92%和52%至61%。因此，这种工程化的Cry2Aa毒素可用于培育抗虫转基因Bt品系，以保护作物免受CLW和CBW等不同害虫造成的损害。
