ABSTRACT

The idea of enhanced methanol production from cell wall by pectin methyl esterase enzymes (PME) combined with expression of cry genes from Bacillus thuringiensis as a strategy to improve insect pest control in cotton is presented. We constructed a cassette containing two cry genes (cry1Fa and Cry32Aa) and two pme genes, one from Arabidopsis thaliana (AtPME), and other from Aspergillus. niger (AnPME) in pCAMBIA1301 plant expression vector using CAMV-35S promoter. This construction was transformed in Eagle-2 cotton variety by using shoot apex-cut Agrobacterium-mediated transformation. Expression of cry genes and pme genes was confirmed by qPCR. Methanol production was measured in control and in the cry and pme transformed plants showing methanol production only in transformed plants, in contrast to the non-transgenic cotton plants. Finally, insect bioassays performed with transgenic plants expressing cry and pme genes showed 100% mortality for Helicoverpa armigera (cotton bollworm) larvae, 70% mortality for Pectinophora gossypiella (pink bollworm) larvae and 95% mortality of Earias fabia, (spotted bollworm) larvae, that was higher than the transgenic plants expressing only cry genes that showed 84%, 49% and 79% mortality, respectively. These results demonstrate that Bt. cry-genes coupled with pme genes are an effective strategy to improve the control of different insect pests.

摘要

提出了通过果胶甲酯酶 (PME) 结合表达苏云金芽孢杆菌cry基因来增强细胞壁甲醇产量的想法，作为改善棉花害虫防治的策略。我们构建了一个包含两个cry基因（cry1Fa和Cry32Aa）和两个pme基因的盒式磁带，一个来自拟南芥（AtPME），另一个来自曲霉。niger ( AnPME ) 在 pCAMBIA1301 植物表达载体中使用 CAMV-35S 启动子。使用茎尖切割农杆菌将该构建体转化到 Eagle-2 棉花品种中-介导的转化。表达一声基因和PME基因通过qPCR证实。在对照和cry和pme转化植物中测量甲醇产量，表明与非转基因棉花植物相比，仅在转化植物中产生甲醇。最后，昆虫生物测定用表达的转基因植物进行的叫声和PME基因显示100％的死亡率为棉铃虫（棉铃虫）幼虫，70％的死亡率为棉红铃虫（棉红铃虫）幼虫和95％的死亡率的Earias法比亚，（斑点棉铃虫）幼虫，这高于仅表达 84%、49% 和 79% 死亡率的仅表达cry基因的转基因植物。这些结果表明 Bt. 将cry基因与pme基因相结合是改善对不同害虫的控制的有效策略。