| 邓宝林，马慧杏，李春燕，李宝琪，刘吉升,2026,细菌侵染后赤拟谷盗幼虫实时荧光定量PCR内参基因的表达稳定性评估[J].环境昆虫学报,(3):837-845 |
| 细菌侵染后赤拟谷盗幼虫实时荧光定量PCR内参基因的表达稳定性评估 |
| Evaluation of reference genes expression stability by quantitative real-time PCR in Tribolium castaneum larvae after bacterial infection |
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| DOI： |
| 中文关键词: 赤拟谷盗 内参基因 实时荧光定量PCR 表达稳定性 |
| 英文关键词:Tribolium castaneum reference genes quantitative real-time PCR expression stability |
| 基金项目:国家自然科学基金（31501898）；广州大学校级大学生创新创业训练项目（XJ202411078128）；广州大学第十九届“挑战杯”学术科技竞赛（2024TZBNAI1609）；第六届广州大学生命科学学院“科创杯”大赛（SKYKCB20240312，SKYKCB20240313） |
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| 中文摘要: |
| 实时定量RT-PCR（qRT-PCR）是进行基因表达水平分析的一种常用技术手段，选择在试验条件下表达相对稳定的内参基因是数据标准化的关键。【目的】本试验旨评估不同细菌侵染后赤拟谷盗Tribolium castaneum幼虫的候选内参基因的表达稳定性，为后续赤拟谷盗的基因表达分析奠定基础。【方法】对赤拟谷盗幼虫进行革兰氏阴性菌大肠杆菌Escherichia coli和革兰氏阳性菌金黄色葡萄球菌Staphylococcus aureus的喂食侵染，通过qRT-PCR检测ACTB、CAD、RpL13A、RpL32、RpS3、RpS6、RpS18和Syx1A等8个候选内参基因在不同组织中和不同侵染时间点的相对表达水平；并利用ΔCq、geNorm、NormFinder和BestKeeper综合评估候选内参基因的表达稳定性。【结果】根据qRT-PCR结果，在不进行细菌侵染的对照组中，RpS3和RpL32是最稳定的内参基因；大肠杆菌侵染时，RpS3和RpL32是最稳定的基因，Syx1A和ACTB是较稳定的基因；金黄色葡萄球菌侵染时，RpS3、RpL32和Syx1A是最稳定的基因。综合结果显示，两种细菌侵染后不同组织中，RpS3和RpL32是表达最稳定的两个基因，而CAD的表达稳定性最差。【结论】在细菌侵染赤拟谷盗幼虫的qRT-PCR试验中，推荐使用RpS3和RpL32作为内参基因，Syx1A和ACTB也可以作为候选内参基因。 |
| 英文摘要: |
| Quantitative real-time PCR (qRT-PCR) is a commonly used technique for analyzing gene expression levels. The selection of a relatively stable reference gene under experimental conditions is crucial for data normalization.【Aim】This study aimed to evaluate the expression stability of candidate reference genes in Tribolium castaneum larvae under different bacterial infections, laying a foundation for subsequent gene expression analysis in T. castaneum.【Methods】Larvae of T. castaneum were infected with Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus by feeding. The relative expression levels of eight candidate reference genes, including ACTB, CAD, RpL13A, RpL32, RpS3, RpS6, RpS18, and Syx1A, were detected by qRT-PCR across different tissues and at various infection time points. The expression stability of these candidate reference genes was comprehensively evaluated using ΔCq, geNorm, NormFinder, and BestKeeper.【Results】Based on qRT-PCR results, RpS3 and RpL32 were identified as the most stable reference genes in the non-infected control group. During E. coli infection, RpS3 and RpL32 remained the most stable genes, while Syx1A and ACTB showed relatively high stability. For S. aureus infection, RpS3, RpL32, and Syx1A were the most stable genes. Comprehensive analysis revealed that RpS3 and RpL32 were the two most stably expressed genes across different tissues and after both bacterial infections, while CAD exhibited the poorest expression stability. 【Conclusion】Therefore, RpS3 and RpL32 are recommended as reference genes for qRT-PCR analysis in the larvae of T. castaneum after bacterial infection. Syx1A and ACTB can also be used as the candidate reference genes. |
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