Cry1Ac的绿色荧光蛋白GFP标记及其在棉铃虫幼虫中肠的定位分布

为了明确Cry1Ac蛋白在棉铃虫体内与中肠组织的相互作用,采用重叠PCR方法将Bt-cry1Ac基因和绿色荧光蛋白GFP基因融合,构建含Cry1Ac毒蛋白和绿色荧光蛋白GFP原核表达载体,并在大肠杆菌大量表达。利用荧光显微镜观察发现,表达Cry1Ac-GFP融合蛋白的大肠杆菌在蓝光激发下发出绿色荧光。将含有融合蛋白的菌液拌入人工饲料饲喂3龄棉铃虫幼虫96h,取棉铃虫幼虫中肠做冰冻切片并在荧光显微镜下观察。结果显示,取食含有Cry1Ac-GFP融合蛋白饲料的棉铃虫幼虫中肠能够发出强烈荧光。比较Cry1Ac杀虫蛋白敏感和抗性棉铃虫幼虫中肠的发光部位,敏感棉铃虫幼虫的中肠围食膜已经消失,肠壁细胞发出强烈的荧光,而抗性棉铃虫的围食膜较健全并发出荧光。

Labeled Bt toxin Cry1Ac protein with green fluorescent protein and its locating and distributing in midgut of larvae of Helicoverpa armigera

In order to exploring the binding of Bt toxin with the midgut of larvae of Helicoverpa armigera Hübner, the fusion gene of cry1Ac and green fluorescent protein (GFP) was constructed by overlap-PCR, and expressed in Escherichia coli. It was found that Cry1Ac-GFP fusion protein expressed in E.coli emit green fluorescence under blue light. Bacteria solution containing Cry1Ac-GFP fusion protein was mixed into artificial diet, on which 3rd instar larvae of H.armigera were raised for 96 hours. After turned to frost slice, midgut was observed under blue light of fluorescence microscope. It was showed that midgut holding Cry1Ac-GFP fusion protein emits strong green fluorescence. Comparing the fluorescence position in midgut of larvae between Bt resistant strain and susceptible strain we found that peritrophic membrane of susceptible strain disappeared, intestinal wall cells was labeled with Cry1Ac-GFP fusion protein and emitted strong fluorescence instead. However, the Cry1Ac-GFP fusion protein only accreted with peritrophic membrane of resistant strain and showed fluorescence, which suggested that the binding of peritrophic membrane with Cry1Ac toxin protein may result in resistance of larvae of H.armigera to Cry1Ac insecti- cidal crystal proteins.