School of Life Sciences, Central China Normal University, Wuhan, 430070, China.
Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, China.
Insect Biochem Mol Biol. 2020 Mar;118:103306. doi: 10.1016/j.ibmb.2019.103306. Epub 2019 Dec 13.
The insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) are powerful tools for insect control. Cry toxin receptors such as cadherin (CAD), ABCC2 transporter and alkaline phosphatase (ALP), located on insect midgut cells, are needed for Cry toxicity. Although insect cell lines are useful experimental models for elucidating toxin action mechanism, most of them show low expression of Cry-receptors genes. The GATA transcription factor family plays important roles in regulating development and differentiation of intestine stem cells. Here, we investigated whether GATAs transcription factors are involved in the expression of Cry1Ac-receptors genes, using multiple insect cell lines. Four GATA genes were identified in the transcriptome of the midgut tissue from the lepidopteran larvae Helicoverpa armigera. These HaGATA genes were transiently expressed in three lepidopteran cell lines, Spodoptera frugiperda Sf9, H. armigera QB-Ha-E5 and Trichoplusia ni Hi5. Analysis of transcription activity using transcriptional gene-fusions showed that only H. armigera GATAe (HaGATAe) significantly increased the transcription of CAD, ABCC2 and ALP receptors genes in all insect cell lines. Key DNA regions for HaGATAe regulation were identified in the promoter sequence of these Cry-receptors genes by using promoter deletion mapping. The transient expression of HaGATAe in these cell lines, conferred sensitivity to Cry1Ac toxin, although in Hi5 cells the susceptibility to Cry1Ac was lower than in other two cell lines. High sensitivity to Cry1Ac correlated with simultaneous transcription of ABCC2 and CAD genes in Sf9 and QB-Ha-E5 cells. Our results reveal that HaGATAe enhances transcription of several lepidopteran Cry1Ac receptor genes in cultured insect cells.
苏云金芽孢杆菌（Bt）产生的杀虫 Cry 毒素是昆虫防治的有力工具。位于昆虫中肠细胞上的 Cry 毒素受体，如钙粘蛋白（CAD）、ABCC2 转运蛋白和碱性磷酸酶（ALP），是 Cry 毒性所必需的。虽然昆虫细胞系是阐明毒素作用机制的有用实验模型，但它们大多数显示 Cry 受体基因表达水平较低。GATA 转录因子家族在调节肠干细胞的发育和分化中发挥重要作用。在这里，我们使用多种昆虫细胞系研究了 GATA 转录因子是否参与 Cry1Ac 受体基因的表达。在鳞翅目幼虫棉铃虫的中肠组织转录组中鉴定出 4 个 GATA 基因。这些 HaGATA 基因在三种鳞翅目细胞系 Spodoptera frugiperda Sf9、H. armigera QB-Ha-E5 和 Trichoplusia ni Hi5 中瞬时表达。使用转录基因融合分析转录活性表明，只有棉铃虫 GATAe（HaGATAe）显着增加了所有昆虫细胞系中 CAD、ABCC2 和 ALP 受体基因的转录。通过启动子缺失作图，在这些 Cry 受体基因的启动子序列中鉴定出 HaGATAe 调节的关键 DNA 区域。HaGATAe 在这些细胞系中的瞬时表达赋予了对 Cry1Ac 毒素的敏感性，尽管在 Hi5 细胞中对 Cry1Ac 的敏感性低于其他两种细胞系。对 Cry1Ac 的高敏感性与 Sf9 和 QB-Ha-E5 细胞中 ABCC2 和 CAD 基因的同时转录相关。我们的结果表明，HaGATAe 增强了培养昆虫细胞中几种鳞翅目 Cry1Ac 受体基因的转录。
