The piggyBac-based germline transformation system was recently established in a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda. Tissue-specific promoters are needed to apply this transformation system to express transgenes in a tissue-specific manner. Highly expressed genes in the midgut were identified by RNA sequencing and RT-qPCR. Promoter regions of 11 genes highly expressed in the midgut were identified and cloned. Baculoviruses expressing the luciferase gene under the control of these promoters were produced and tested in the FAW. These baculoviruses did not show significant luciferase activity in the FAW midgut. Four transgenic FAW lines expressing the luciferase gene under the control of the SfSP38/P2000, SfCalphotin/P2000, SfMG17/P2000, and SfCPH38/P2000 promoters were generated using piggyBac-based germline transformation methods. Significantly higher luciferase activity was detected in the midgut than in other tissues of transgenic FAW. The SfCPH38/P2000 promoter with the highest activity and midgut specificity was used to drive the expression of P450, SfCYP321A8, which is known to be involved in deltamethrin resistance. Higher mRNA levels of SfCYP321A8 and P450 activity were detected in the midgut of transgenic larvae than in wild-type larvae. Bioassays showed that transgenic larvae expressing SfCYP321A8 in the midgut are tolerant to deltamethrin. Here, we presented methods for the identification of midgut-specific promoters in the FAW and used them to study the role of P450 overexpression in the midgut on insecticide resistance. These methods could also be used to identify other tissue-specific promoters for applications of piggyBac-based germline transformation in functional genomics in FAW and other non-model insects.

Graphical abstract

中文翻译：

---

CYP321A8 P450基因的中肠特异性表达增加秋粘虫草地夜蛾对溴氰菊酯的耐受性

基于piggyBac的种系转化系统最近在全球农业害虫秋粘虫 (FAW)、草地贪夜蛾中建立. 需要组织特异性启动子来应用这种转化系统以组织特异性方式表达转基因。通过 RNA 测序和 RT-qPCR 鉴定了中肠中高表达的基因。鉴定并克隆了在中肠中高度表达的11个基因的启动子区域。在这些启动子的控制下表达荧光素酶基因的杆状病毒在 FAW 中产生和测试。这些杆状病毒在 FAW 中肠中没有显示出显着的荧光素酶活性。使用piggyBac产生了在 SfSP38/P2000、SfCalphotin/P2000、SfMG17/P2000 和 SfCPH38/P2000 启动子控制下表达荧光素酶基因的四种转基因 FAW 系种系转化方法。在中肠中检测到显着高于转基因FAW的其他组织的荧光素酶活性。具有最高活性和中肠特异性的 SfCPH38/P2000 启动子用于驱动已知与溴氰菊酯抗性有关的 P450、 SfCYP321A8的表达。更高的SfCYP321A8 mRNA 水平与野生型幼虫相比，在转基因幼虫的中肠中检测到 P450 和 P450 活性。生物测定表明，在中肠中表达 SfCYP321A8 的转基因幼虫对溴氰菊酯具有耐受性。在这里，我们提出了在 FAW 中鉴定中肠特异性启动子的方法，并用它们来研究 P450 在中肠过表达对杀虫剂抗性的作用。这些方法还可用于鉴定其他组织特异性启动子，以将基于piggyBac的种系转化应用于 FAW 和其他非模型昆虫的功能基因组学中。

图形概要