Effect Of Cry1Ac Toxin From Bacillus Thuringiensis On APNs Expression In Midgut Of Helicoverpa Armigera

The insecticidal crystal proteins of Bt are widely used for crop pest control. H.armigera used as the model insect for Bt studies, is an improtant widespread and polyphagous agriculture pest, which is sensitive to Cry toxins. Bt toxin proteins play a vital role in pest control, but if the insects are under Bt toxin environment for a long time, the resistant problems appears.In this study, we used the midguts of4th sensitive, resistant and Cry1Ac toxin treated sensitive H.armigera larvae as research material and set the four aminopeptidase-Ns (apnl, apn2, apn4and apn5)) in the midgut of H.armigera larvae, which were cloned based on early mass spectrometry data of our laboratory, as study objects. In order to detect the effects of Cry1Ac on the transcription levels and translation levels of the four APNs, Real-time quantitative polymerase chain reaction (qRT-PCR), Western blot and immunohistochemistry analysis (DAB immunohistochemistry and fluorescence immunohistochemistry) were used. And we also observe and compare the distribution and expression levels of four APNs in midguts between the sensitive larvae and resistant ones by laser scanning confocal microscope. The main results are as follows:1. The effect of Cry1Ac on apns transcription levels:The results of RT-qPCR show that the transcription level of apnl gene was the highest among the four genes, followed by apn2, apn5, and apn4was the lowest one. After treated with CrylAc toxins for36hours, transcription levels of the four apns were all up-regulated in defferent degrees.2. The effect of Cry1Ac on APNs translation levels:The results of Western blot analysis, DAB immunohistochemistry and fluorescence immunohistochemistry indicate that the protein level of APN1was the highest and the APN4was the lowest. After toxin treating, the four APNs protein levels were up-regulated, which is in line with mRNA levels. According to the immunohistochemistry analysis, we can direct see that all the four APNs were evenly distributed in the epithelial cell of midgut. In addition, we found a sharp down-regulation of ALP after toxin treatment by SDS-PAGE/LC-MS. APN activities detection demonstrates that the total APN activities in toxin treating midgut increased by16.8%.3. Defferent expression of the four APNs between sensitive insects and resistant insects:Fluorescence immunohistochemistry observed by laser scanning confocal microscope show the four APNs distributions in midguts of sensitive larvae were similar with their in resistant larvae, which were all present in the epithelial cell of midgut. The amounts of APN1and APN2in midgut of sensitive insects larvae were much more than that in resistant ones, but the amounts of APN4and APN5in midgut of sensitive insects larvae were a little less than that in resistant ones.With toxin treatment, there appeared up-regulations of APNs and down-regulation of alkaline phosphatase(ALP), and it may be the mechanisms of instant reaction and self-protection caused by Cry toxin. The down-regulation of APN1and APN2in midgut of resistant larvae suggested that APN1and APN2may be the receptors of Cry1Ac and the critical factors of resistance to Cry1Ac, but APN4and APN5may not be the receptors of Cry1Ac and may have a main role of detoxication and self-protection when the larvae in toxic environment, which need further researches. All the study results will provide theoretical basis for the further research on efficient insecticidal biopesticide study and resistant mechanism revealing to slow down or eliminate insects resistance to Bt toxin.