Evaluation of candidate reference genes for gene expression analysis in the brassica leaf beetle, Phaedon brassicae (Coleoptera: Chrysomelidae).
Evaluation of candidate reference genes for gene expression analysis in the brassica leaf beetle, Phaedon brassicae (Coleoptera: Chrysomelidae).
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DOI：
10.1371/journal.pone.0251920
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Zhang W
中科院分区：
文献类型：
--
作者：
Ma L;Jiang T;Liu X;Xiao H;Peng Y;Zhang W
芸苔叶甲虫 Phaedon bassicae 是一种臭名昭著的十字花科蔬菜落叶害虫。然而，由于序列数据有限，对该害虫的分子研究很少。最近，RNA 测序提供了一个强大的平台来生成大量转录组数据，这需要 RT-qPCR 来验证目标基因的表达。选择可靠的内参基因来标准化 RT-qPCR 数据是基因表达分析的先决条件。在本研究中，使用四种不同的统计算法评估了八个候选参考基因在生物条件（发育阶段和各种组织）和非生物扰动（热应激和农药暴露）下的表达稳定性。针对各自的实验条件推荐了最佳的参考基因套件。对于组织表达分析，推荐RPL32和EF-1α作为合适的内参基因。 RPL19和TBP是不同发育阶段的最佳内参基因。 RPL32 和 TBP 被确定为最合适的热应力参考。此外，RPL32 和 RPL19 被列为杀虫剂暴露的最佳参考。这项工作对各自实验条件下的最佳参考基因进行了系统探索，我们的发现将促进甘蓝的分子研究。
The brassica leaf beetle Phaedon brassicae is a notorious defoliator of cruciferous vegetables. However, few molecular studies of this pest have been conducted due to limited sequence data. Recently, RNA sequencing has offered a powerful platform to generate numerous transcriptomic data, which require RT-qPCR to validate target gene expression. The selection of reliable reference genes to normalize RT-qPCR data is a prerequisite for gene expression analysis. In the present study, the expression stabilities of eight candidate reference genes under biotic conditions (development stages and various tissues) and abiotic perturbations (thermal stress and pesticide exposure) were evaluated using four different statistical algorithms. The optimal suites of reference genes were recommended for the respective experimental conditions. For tissue expression analysis, RPL32 and EF-1α were recommended as the suitable reference genes. RPL19 and TBP were the optimal reference genes across different developmental stages. RPL32 and TBP were identified as the most suitable references for thermal stress. Furthermore, RPL32 and RPL19 were ranked as the best references for insecticide exposure. This work provides a systematic exploration of the optimal reference genes for the respective experimental conditions, and our findings would facilitate molecular studies of P. brassicae.
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