Abstract
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.
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Acknowledgments
We thank Jeff Howell from the University of Kentucky for help with FAW rearing, Dr. Anjiang Tan of the Shanghai Institute of Plant Physiology and Ecology, China, for the gift of the piggyBac vector, and Dr. Cynthia L. Goodman from USDA, ARS, BCIRL at Columbia, MO, for the gift of SfMG_0617 cells. This material is based on work supported by the National Science Foundation I/UCRC, the Center for Arthropod Management Technologies, under Grant No. IIP1821936, industry partners, and the National Institute of Food and Agriculture, US Department of Agriculture (under HATCH Project 2353057000).
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Xien Chen and Subba Reddy Palli designed and conceived the study. Xien Chen performed experiments. Xien Chen and Subba Reddy Palli analyzed data and wrote the manuscript.
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Chen, X., Palli, S.R. Midgut-specific expression of CYP321A8 P450 gene increases deltamethrin tolerance in the fall armyworm Spodoptera frugiperda. J Pest Sci 96, 1611–1623 (2023). https://doi.org/10.1007/s10340-022-01483-7
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DOI: https://doi.org/10.1007/s10340-022-01483-7