School of Life Sciences, Central China Normal University, Wuhan 430070, China; Applied Biotechnology Center, Wuhan University of Bioengineering, Wuhan 430415, China.
Institute of Hubei Agriculture Academy, Wuhan 430070, China.
Pestic Biochem Physiol. 2023 Aug;194:105516. doi: 10.1016/j.pestbp.2023.105516. Epub 2023 Jul 7.
Helicoverpa armigera is a worldwide pest that has been efficiently controlled by transgenic plants expressing Bt Cry toxins. To exert toxicity, Cry toxins bind to different receptors located in larval midgut cells. Previously, we reported that GATA transcription factor GATAe activates the expression of multiple H. armigera Cry1Ac receptors in different insect cell lines. Here, the mechanism involved in GATAe regulation of HaABCC2 gene expression, a key receptor of Cry1Ac, was analyzed. HaGATAe gene silencing by RNAi in H. armigera larvae confirmed the activation role of HaGATAe on the expression of HaABCC2 in the midgut. The contribution of all potential GATAe-binding sites was analyzed by site-directed mutagenesis using Hi5 cells expressing a reporter gene under regulation of different modified HaABCC2 promoters. DNA pull-down assays revealed that GATAe bound to different predicted GATA-binding sites and mutations of the different GATAe-binding sites identified two binding sites responsible for the promoter activity. The binding site B9, which is located near the transcription initiator site, has a major contribution on HaABCC2 expression. Also, DNA pull-down assays revealed that all other members of GATA TF family in H. armigera, besides GATAe, HaGATAa, HaGATAb, HaGATAc and HaGATAd also bound to the HaABCC2 promoter and decreased the GATAe dependent promoter activity. Finally, the potential participation in the regulation of HaABCC2 promoter of several TFs other than GATA TFs expressed in the midgut cells was analyzed. HaHR3 inhibited the GATAe dependent activity of the HaABCC2 promoter, while two other midgut-related TFs, HaCDX and HaSox21, also bound to the HaABCC2 promoter region and increased the GATAe dependent promoter activity. All these data showed that GATAe induces HaABCC2 expression by binding to HaGATAe binding sites in the promoter region and that additional TFs participate in modulating the HaGATAe-driven expression of HaABCC2.
棉铃虫是一种世界性的害虫，其生长受到转 BtCry 毒素基因植物的有效控制。为了发挥毒性，Cry 毒素与位于幼虫中肠细胞中的不同受体结合。此前，我们报道 GATA 转录因子 GATAe 激活了不同昆虫细胞系中多种棉铃虫 Cry1Ac 受体的表达。在这里，分析了 GATAe 调节 HaABCC2 基因表达的机制，HaABCC2 是 Cry1Ac 的一个关键受体。通过棉铃虫幼虫中的 RNAi 沉默 HaGATAe 基因，证实了 HaGATAe 对中肠中 HaABCC2 表达的激活作用。通过使用表达受不同修饰的 HaABCC2 启动子调控的报告基因的 Hi5 细胞，对所有潜在的 GATAe 结合位点进行了定点突变分析。DNA 拉下测定表明，GATAe 与不同预测的 GATA 结合位点结合，并且鉴定出不同 GATAe 结合位点的突变导致两个负责启动子活性的结合位点。位于转录起始位点附近的结合位点 B9 对 HaABCC2 表达有主要贡献。此外，DNA 拉下测定还表明，在棉铃虫中，除 GATAe 外，GATAa、GATAb、GATAc 和 GATAd 等 GATA TF 家族的所有其他成员也与 HaABCC2 启动子结合，并降低了 GATAe 依赖性启动子活性。最后，还分析了中肠细胞中表达的除 GATA TF 之外的几种 TF 对 HaABCC2 启动子的潜在调节作用。HaHR3 抑制了 HaABCC2 启动子的 GATAe 依赖性活性，而另外两个与中肠相关的 TF，HaCDX 和 HaSox21，也与 HaABCC2 启动子区域结合，并增加了 GATAe 依赖性启动子活性。所有这些数据表明，GATAe 通过与启动子区域中的 HaGATAe 结合位点结合来诱导 HaABCC2 的表达，并且其他 TF 参与调节 HaGATAe 驱动的 HaABCC2 表达。
