Midgut-specific expression of CYP321A8 P450 gene increases deltamethrin tolerance in the fall armyworm Spodoptera frugiperda
Midgut-specific expression of CYP321A8 P450 gene increases deltamethrin tolerance in the fall armyworm Spodoptera frugiperda
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DOI：
10.1007/s10340-022-01483-7
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发表时间：
2022-02-16
影响因子：
4.8
通讯作者：
Palli，Subba Reddy
中科院分区：
文献类型：
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作者：
Chen，Xien;Palli，Subba Reddy
以全球性农业害虫草地贪夜蛾（Spodoptera frugiperda）为研究对象，建立了基于piggyBac的转基因体系。组织特异性启动子是应用该转化系统以组织特异性方式表达转基因所必需的。通过RNA测序和RT-qPCR鉴定中肠中的高表达基因。鉴定并克隆了中肠高表达的11个基因的启动子区。在FAW中产生并测试了在这些启动子控制下表达荧光素酶基因的杆状病毒。这些杆状病毒在FAW中肠中没有显示出显著的荧光素酶活性。利用piggyBac的胚系转化方法，获得了4个在SfSP 38/P2000、SfCalphotin/P2000、SfMG 17/P2000和SfCPH 38/P2000启动子控制下表达荧光素酶基因的转基因FAW株系。在中肠中检测到的荧光素酶活性显著高于转基因FAW的其他组织。具有最高活性和中肠特异性的SfCPH 38/P2000启动子用于驱动P450、SfCYP 321 A8的表达，已知其参与溴氰菊酯抗性。转基因幼虫中肠SfCYP 321 A8和P450活性的mRNA水平高于野生型幼虫。生物测定表明，在中肠中表达SfCYP 321 A8的转基因幼虫对溴氰菊酯具有耐受性。在这里，我们提出了方法，用于识别中肠特异性启动子在FAW和使用它们来研究的作用，P450过表达的中肠杀虫剂抗性。这些方法也可用于鉴定其他组织特异性启动子，为基于piggyBac的生殖系转化在FAW和其他非模式昆虫功能基因组学中的应用奠定基础。
ThepiggyBac-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 usingpiggyBac-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 ofSfCYP321A8and 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 ofpiggyBac-based germline transformation in functional genomics in FAW and other non-model insects.Graphical abstract