Balakrishnan Indumathi Kamatchi, Dubey Himanshu, Shukla Pawan, Debnath Rajal, Vanitha C, Subrahmanyam Gangavarapu, Moorthy Manthira, Arunkumar Kallare P, Kumar Vikram, Singh Abhishek, Ponnuvel K M
Seribiotech Research Laboratory, Central Silk Board, Kodathi, Bangalore, India.
Department of Biotechnology, School of Sciences, Jain University, Bengaluru, India.
Mol Biol Rep. 2025 Jul 2;52(1):668. doi: 10.1007/s11033-025-10787-7.
Gene expression profiling is vital for deciphering immune responses in insects, especially for genes involved in pathogen defense, stress adaptation, and immune regulation. The muga silkworm, Antheraea assamensis Helfer (A. assamensis), a species of considerable economic and cultural significance in Northeast India, is vulnerable to bacterial and viral infections that severely impact silk production. Despite its importance, no prior studies have validated suitable reference genes for reverse-transcription quantitative PCR (RT-qPCR) based expression analysis under pathogenic stress in this species. Given that reference gene expression may vary with tissue type, developmental stage, and infection status, rigorous validation of stable reference genes is essential for accurate normalization.
Eight candidate housekeeping genes (EF1α, GAPDH, RPS3A, RPL13A, Actin-A1, Tub1, SDHA, and EIF4A) were selected for evaluation. RT-qPCR assays were conducted on fat body and midgut tissues under bacterial and viral infection conditions. Gene expression stability was assessed using the geNorm, NormFinder, and RefFinder algorithms. Results indicated that GAPDH was the most stable gene in the fat body during bacterial infection, whereas Actin-A1 and RPS3A exhibited the highest stability under viral infection. In the midgut, GAPDH showed the highest stability during bacterial infection, while both GAPDH and Tub1 were stable under viral infection conditions. Overall, RPS3A demonstrated the most consistent expression stability across all tissues and infection conditions.
This study provides the first validated reference gene set for A. assamensis under pathogenic stress, highlighting RPS3A as the most reliable gene for RT-qPCR normalization. These findings will improve the accuracy of gene expression studies and support future transcriptomic and functional genomic research in this economically important silkmoth species.
基因表达谱分析对于解读昆虫的免疫反应至关重要，特别是对于参与病原体防御、应激适应和免疫调节的基因。阿萨姆柞蚕（Antheraea assamensis Helfer，简称A. assamensis）是印度东北部具有重要经济和文化意义的物种，易受细菌和病毒感染，这严重影响了丝绸生产。尽管其具有重要性，但此前尚无研究验证该物种在致病应激下基于逆转录定量PCR（RT-qPCR）的表达分析的合适内参基因。鉴于内参基因的表达可能因组织类型、发育阶段和感染状态而异，严格验证稳定的内参基因对于准确标准化至关重要。
选择了八个候选管家基因（EF1α、GAPDH、RPS3A、RPL13A、Actin-A1、Tub1、SDHA和EIF4A）进行评估。在细菌和病毒感染条件下，对脂肪体和中肠组织进行了RT-qPCR分析。使用geNorm、NormFinder和RefFinder算法评估基因表达稳定性。结果表明，在细菌感染期间，GAPDH是脂肪体中最稳定的基因，而Actin-A1和RPS3A在病毒感染下表现出最高的稳定性。在中肠中，GAPDH在细菌感染期间表现出最高的稳定性，而在病毒感染条件下，GAPDH和Tub1均稳定。总体而言，RPS3A在所有组织和感染条件下表现出最一致的表达稳定性。
本研究为致病应激下的阿萨姆柞蚕提供了首个经过验证的内参基因集，突出了RPS3A作为RT-qPCR标准化最可靠基因的地位。这些发现将提高基因表达研究的准确性，并支持对这种具有重要经济意义的蚕蛾物种未来的转录组学和功能基因组学研究。
