Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae).
Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae).
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DOI：
10.1371/journal.pone.0181862
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Du YZ
中科院分区：
文献类型：
--
作者：
Chang YW;Chen JY;Lu MX;Gao Y;Tian ZH;Gong WR;Zhu W;Du YZ
三叶斑潜蝇（Liriomyza trifolii）是一种高度入侵的采叶昆虫，在世界范围内对蔬菜和园艺作物造成严重损害。相对较少的研究定量的基因表达L。采用实时定量PCR（RT-qPCR）检测三叶草中的基因表达，这是一种可靠且灵敏的检测基因表达的技术。RT-qPCR需要选择参考基因以标准化基因表达数据并控制样品之间的内部差异。本研究中，9个管家基因从L。根据它们在使用geNorm、Normfinder、BestKeeper、ΔCt方法和Refl.将编码热休克蛋白21.7的HSP 21.7作为靶基因，以验证候选参考基因的表达。结果表明，ACTIN和18 S基因最适合在发育阶段和低温下表达，TUB和18 S基因在性别上表达最稳定，GAPDH和ACTIN基因是高温下基因表达监测的最佳参考基因。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，这提高了表达数据的准确性和质量。本研究结果为建立标准化的RT-qPCR方法提供了参考基因的信息，对开展乳杆菌功能基因组学研究具有重要的参考价值。三叶草。
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.
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DOI：
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