基于RNAi沉默与转录组分析的埃及伊蚊AaTryp1基因功能解析*
Functional analysis of the Aedes aegypti AaTryp1 gene based on RNAi and transcriptomics
段顶峰1, 2** 龚 雪1, 2 邬溢杨1, 2 余忠浩1, 2 周艳娇1, 2 聂萌1, 2 廖承红1, 2***
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DOI：10.7679/j.issn.20951353.2026.111
作者单位：1. 海南大学生命健康学院病媒生物学实验室，海南省全健康重点实验室，海南省协同创新中心，海口 570228； 2. 海南大学国际全健康研究院，海口 570228
中文关键词：AaTryp1；RNA干扰；转录组；差异表达基因
英文关键词：AaTryp1; RNAi; transcriptome; differentially expressed genes
中文摘要：【目的】 明确埃及伊蚊Aedes aegypti胰蛋白酶 1 基因（AaTryp1）的组织及发育阶段表达特征，通过RNA干扰（RNA interference，RNAi）结合转录组测序（RNA-seq）技术解析该基因沉默后的分子响应及潜在生物学功能，为深入阐明其在蚊虫生理调控中的作用机制提供理论依据。【方法】 采用RT-qPCR技术检测AaTryp1在埃及伊蚊不同组织、发育阶段的表达差异；通过体外合成靶向AaTryp1的dsRNA并导入雌蚊体内，结合RNA-seq技术系统分析基因沉默后的转录组变化，对筛选获得的差异表达基因（Differentially expressed genes，DEGs）进行GO（Gene Ontology）功能富集及KEGG（Kyoto Encyclopedia of Genes and Genomes）通路分析，并对干扰后DEGs进行定量验证。【结果】 AaTryp1在埃及伊蚊雌蚊中呈现特异性高表达，其中唾液腺、腿部及中肠组织的表达量显著（P < 0.05）高于其他组织，且吸血前后表达水平存在显著差异；RNAi处理后，AaTryp1被有效敲低，转录组分析共筛选出110个DEGs。GO富集分析显示，这些DEGs主要富集于代谢过程、细胞过程等生物学过程，以及催化活性、结合活性等分子功能类别；KEGG通路分析表明，DEGs显著富集于肾素-血管紧张素系统、神经活性配体-受体相互作用、脂肪酸生物合成等非经典消化与免疫相关通路。RT-qPCR验证结果与RNA-seq表达趋势一致，证实测序数据可靠。【结论】 AaTryp1在埃及伊蚊雌蚊中具有组织特异性和吸血响应性表达特征，其功能不仅可能涉及经典的消化代谢过程，还可能参与体液稳态调节、神经信号传导及脂质代谢等多个关键生理过程。本研究为后续深入解析该基因的调控机制及蚊虫防控靶标的开发提供了重要基础。
英文摘要：[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.