TmTRAF acts as a negative regulator of antimicrobial peptide production against microbes in Tenebrio molitor = 갈색거저리에서 미생물에 대한 항균펩타이드 생산의 음성 조절 기능을 하는 TmTRAF

다국어 초록 (Multilingual Abstract)

Tumor necrosis factor receptor-associated factor (TRAF) is an adaptor protein that mediates signal transduction in the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) pathway of mammals, playing crucial role in regulating innate immunity and inflammatory responses. However, the function of TRAF in the Toll signaling pathway of insects remains unclear. In this study, the TRAF identified from the Tenebrio molitor nucleotide database was found to contain conserved RING and MATH domains and to share phylogenetic similarity with insect (Coleoptera, Diptera, Lepidoptera, Hymenoptera, Hemiptera) TRAF6. Following systemic microbial infection with Escherichia coli, Staphylococcus aureus, and Candida albicans, the temporal and spatial expression patterns of TmTRAF were investigated at 3-, 6-, 9-, 12-, and 24-hours post-infection in young larvae. Induced expression was observed across all tested tussues, suggesting its involvement in both systemic and local immune responses. To investigate the immunological function of TmTRAF in response to microbial challenge, the expression of antimicrobial peptide (AMP) genes and larval survival rates were examined in TmTRAF-silenced larvae. Unexpectedly, TmTRAF knockdown resulted in a significant increase in AMP expression and improved larval survival compared to the control group, a finding contradictory to the established positive regulatory role of TRAF6 reported in other species. These result suggest that TmTRAF acts as a negative regulator, modulating the balance between the Toll and Imd signaling pathways in T. molitor. Collectively, this study provides the first evidence for an inhibitory role of TRAF in insects, thereby offering new insights into the evolutionary diversity and homeostatic regulation of insect immune signaling.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 5
• 2.1. Insect rearing 5
• 2.2. In silico analysis of TmTRAF 5
• 2.3. Full-length cDNA cloning of TmTRAF 6
• 2.3.1. Insert DNA PCR 6
• 2.3.2. Ligation & Transformation 6
• 2.4. Preparation of microorganism 8
• 2.5. RNA extraction and cDNA synthesis 9
• 2.6. Expression and induction patterns of TmTRAF 9
• 2.7. qRT-PCR 10
• 2.8. dsRNA synthesis and RNAi efficiency 11
• 2.9. Mortality 12
• 2.10. Expression of AMPs and NF-κB genes 13
• 2.11. Statistical analysis 14
• Ⅲ. Results 17
• 3.1. In silico characterization of TmTRAF 17
• 3.2. Developmental stage and tissue-specific expression patterns of TmTRAF 22
• 3.3. Induction patterns of TmTRAF in the whole body and tissues after microbial challenge 25
• 3.4. Effects of TmTRAF knockdown on larval survival 27
• 3.5. Effects of TmTRAF RNAi on antimicrobial peptide expression 29
• 3.6. Effects of TmTRAF RNAi on NF-κB gene expression 43
• Ⅳ. Discussion 47
• Ⅴ. Reference 55