Functional analysis of the Aedes aegypti AaTryp1 gene based on RNAi and transcriptomics
Author of the article:DUAN Ding-Feng1, 2** GONG Xue1, 2 WU Yi-Yang1, 2 YU Zhong-Hao1, 2 ZHOU Yan-Jiao1, 2 NIE Meng1,
Author's Workplace：1. Hainan Provincial Collaborative Innovation Center, Hainan Provincial Key Laboratory of One Health, Laboratory of Medical Entomology, School of Life and Health Sciences, Hainan University, Haikou 570228, China; 2. International One Health Research Institute, Hainan University, Haikou 570228, China
Key Words：AaTryp1; RNAi; transcriptome; differentially expressed genes
Abstract：[Aim] To characterize differences in the expression patterns of the trypsin 1 gene of Aedes aegypti (AaTryp1) in different tissues and developmental stages, and to investigate the molecular response and potential biological functions of this gene by combining RNA interference (RNAi) with transcriptome sequencing (RNA-seq), thereby providing a theoretical basis for elucidating its physiological role in mosquitoes. [Methods] RT-qPCR was used to determine the differences in the expression of AaTryp1 in different tissues and developmental stages. Double-stranded RNA targeting AaTryp1 was synthesized in vitro and introduced into female mosquitoes. RNA-seq was then performed to systematically analyze transcriptomic changes following gene silencing. The identified differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and selected DEGs were further validated using quantitative real-time PCR. [Results] AaTryp1 showed tissue-specific expression in female A. aegypti, with significantly higher expression in the salivary glands, legs and midgut, than in other tissues. Its expression differed significantly before and after a blood meal. RNAi treatment effectively knocked down AaTryp1. Transcriptome analysis identified 110 DEGs. GO enrichment analysis showed that these DEGs were mainly associated with metabolic and cellular processes, as well as molecular functions including catalytic activity and binding. KEGG pathway analysis revealed significant enrichment in several non-classical digestion-and immunity-related pathways, including the renin-angiotensin system, neuroactive ligand-receptor interaction and fatty acid biosynthesis. RT-qPCR validation was consistent with the RNA-seq results, confirming the reliability of the sequencing data. [Conclusion] AaTryp1 exhibits tissue-specific, blood-meal related, expression in female A. aegypti. Its function may not be limited to classical digestive metabolism, but may also be involved in hemolymph homeostasis, neural signaling and lipid metabolism. This study provides an important foundation for further elucidation of the regulatory mechanism of AaTryp1, and for the development of novel mosquito control targets.