| Crystal toxin protein,produced by Bacillus thuringiensis(Bt)or Bt-transgenic plants,is the most widely used as green biocontrol and insecticidal microbial component.The Bt toxin receptors,such as cadherin(CAD),ABCC2 transporter(ABCC2)and alkaline phosphatase(ALP)which localized on the brush border membrane of insect midgut epithelial cells,are necessary for the virulence of crystal toxin.Mutation or down-regulation of Bt toxin receptors genes is one of the important resistance mechanisms of insect to Bt crystal toxin protein.Cell lines are important tools to study the regulation mechanism of gene expression,but Bt toxin receptors genes are not expressed or at a low level in the established insect cell lines,which seriously hinders the study of the regulation mechanism of Bt toxin receptor gene expression.The expression of dominant regulatory genes mediated by transfection can generally form a new regulatory network system of transcription factors in cells,thus changing the characteristics of cells.Therefore,this study will screen the important transcription factors that can activate or promote the expression of Bt toxin receptors genes in insect cell lines,in order to research its regulation mechanism on Bt toxin receptor gene expression.The study will provide an important reference for the research on the resistance mechanism of insect to Bt.The GATA transcription factor family as a chromatin remodeling factor has been reported to play an important role in regulating the development and differentiation of intestinal stem cells and so on.Therefore,we first used a variety of insect cell lines to investigate whether members of the GATA transcription factor family in Helicoverpa armigera can regulate the expression of Bt Cry1Ac receptors genes.Five GATA protein genes(GATAa,b,c,d,e)were identified and cloned according to the transcriptome data from Helicoverpa armigera midgut tissue.Bioinformatics analysis of these five GATA proteins showed that all of them had zinc finger structures and contained 1～2 different motifs of GATA binding sites.When HaGATAs were transiently expressed in three different insect cell lines,the results showed that only HaGATAe protein significantly up-regulated the activities of CAD,ABCC2 and ALP promoters of Bt toxin receptors genes in Helicoverpa armigera,and the increased levels ranged from 4 to 857 times.The Highest expression of the three promoters was observed in Sf9 cells compared with Ha or Hi5 cells.In addition,the transcription levels of endogenous ABCC2 gene were significantly enhanced in Ha(37 fold)and Sf9(57 fold)cells with transient expression of HaGATAe,and the transcription of CAD was enhanced in all the three cell lines.Consistently,the susceptibility of Sf9 cells and Ha cells to Cry1Ac toxin greatly increased,with the EC50 of activated Cry1Ac toxin was 0.46 μg/ml and 1.17 μg/ml,respectively.RNAi of HaGATAe could decrease the expression of HaABCC2,HaCAD and other toxin receptors genes in midgut tissue of Helicoverpa armigera larvae.Based on the research above,we also analyzed the core regions of the receptors genes promoters interacting with the HaGATAe.By the truncation of Cry1Ac-receptor promoters,co-transfection and dual luciferase reporter system,the key regions of the promoters were identified as follows:HaABCC2 promoter,-201 bp～-40 bp;HaCAD promoter,-129 bp～-12 bp;HaALP1 promoter,-217 bp～-117 bp.There were 9 potential GATA binding sites(PGBS)in the key promoter region of HaABCC2,five PGBS in HaCAD promoter and one PGBS in HaALP1 promoter.Using the methods of DNA Pull-down and dual-luciferase report assay,it was further confirmed the binding sites of HaGATAe in the key region of the HaABCC2 promoter and its regulation of promoter activity.The results showed that HaGATAe could bind to the-201 bp～-40 bp region of HaABCC2 promoter.Next,a series of DNA fragments with one or multiple GATA binding sites through mutation of other sites were constructed.The DNA pull-down in vitro assay showed HaGATAe factor could bind to multiple GATA binding sites in the key region of the HaABCC2 promoter.The mutation of single site had less influence on activity of the promoter except one site near the initiator site of transcription.Mutation of dual sites showed only two dual sites might inhibit the in vitro activity of the promoter.All the mutations of three to five sites made the activity of the promoter significantly decrease.Finally,we analyzed the regulation of HaGATAe-dependent ABCC2 promoter activity by several other transcription factors.When the plasmid expressing GATAe was co-transfeced with other GATAs into Sf9 cells,the results of DNA Pull-down and dual-luciferase report assay revealed that GATAa,b,c and d could also bind to the core region of the promoter of HaABCC2 gene and inhibit the activity of HaABCC2 promoter induced by GATAe.The efects of FoxA,Sox21 and CDX1 on the activities of GATAe-dependent promoters were also analyzed.Transient expression of the pioneer transcription factor FoxA can reduce the expression of recombinant HaGATAe and thus partially down-regulate the expression level of HaABCC2 receptor gene induced by HaGATAe.Sox21 and CDX1 also bound to the key region of the HaABCC2 promoter and upregulated GATAe-dependent HaABCC2 promoter activity.In conclusion,HaGATAe could up-regulate the expression levels of multiple Bt toxin receptors genes in Helicoverpa armigera.HaGATAe factor could directly bind to multiple binding sites in the key region of the HaABCC2 promoter.Mutations at a single binding site had no or less influence on the activity of the promoter,while mutations at multiple binding sites significantly affected the activity of the promoter.In addition,FoxA,Sox21 and CDX1 also co-regulated HaGATAe-dependent HaABCC2 promoter activity.In this study,we established an experimental system to study the regulation of Bt toxin receptosr genes expression,analyzed the mechanism of regulation of HABCC2 expression.These findings have provided an important reference for exploring the mechanism of insect resistance resulting from the down-regulation of receptor gene expression,and they are conducive to monitoring and management of insect resistance to Bt. |
