Chang Ya-Wen, Chen Jing-Yun, Lu Ming-Xing, Gao Yuan, Tian Zi-Hua, Gong Wei-Rong, Zhu Wei, Du Yu-Zhou
School of Horticulture and Plant Protection & Institute of Applied Entomology, Yangzhou University, Yangzhou, China.
Laboratory for Prevention and Control of Alien Pests, Suzhou Entry-Exit Inspection and Quarantine Bureau, Suzhou, China.
PLoS One. 2017 Jul 26;12(7):e0181862. doi: 10.1371/journal.pone.0181862. eCollection 2017.
Liriomyza trifolii is a highly-invasive leafmining insect that causes significant damage to vegetables and horticultural crops worldwide. Relatively few studies have quantified gene expression in L. trifolii using real-time quantitative PCR (RT-qPCR), which is a reliable and sensitive technique for measuring gene expression. RT-qPCR requires the selection of reference genes to normalize gene expression data and control for internal differences between samples. In this study, nine housekeeping genes from L. trifolii were selected for their suitability in normalizing gene expression using geNorm, Normfinder, BestKeeper, the ΔCt method and RefFinder. HSP21.7, which encodes heat shock protein 21.7, was used as a target gene to validate the expression of candidate reference genes. Results indicated that ACTIN and 18S were optimal for developmental stage and low temperature, TUB and 18S showed the most stable expression for sex, and GAPDH and ACTIN were the best reference genes for monitoring gene expression at high temperature. Selection and validation of appropriate reference genes are critical steps in normalizing gene expression levels, which improve the accuracy and quality of expression data. Results of this study provide vital information on reference genes and is valuable in developing a standardized RT-qPCR protocol for functional genomics research in L. trifolii.
三叶斑潜蝇是一种极具入侵性的潜叶昆虫，对全球范围内的蔬菜和园艺作物造成了严重损害。相对较少的研究使用实时定量PCR（RT-qPCR）对三叶斑潜蝇的基因表达进行定量，RT-qPCR是一种测量基因表达的可靠且灵敏的技术。RT-qPCR需要选择参考基因来标准化基因表达数据并控制样本间的内在差异。在本研究中，从三叶斑潜蝇中选择了9个管家基因，利用geNorm、Normfinder、BestKeeper、ΔCt法和RefFinder评估它们在标准化基因表达方面的适用性。编码热休克蛋白21.7的HSP21.7用作靶基因，以验证候选参考基因的表达。结果表明，ACTIN和18S在发育阶段和低温条件下是最佳参考基因，TUB和18S在性别方面表现出最稳定的表达，而GAPDH和ACTIN是监测高温下基因表达的最佳参考基因。选择和验证合适的参考基因是标准化基因表达水平的关键步骤，可提高表达数据的准确性和质量。本研究结果提供了有关参考基因的重要信息，对于开发用于三叶斑潜蝇功能基因组学研究的标准化RT-qPCR方案具有重要价值。
