Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, Zhejiang, China.
Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, Hangzhou, Zhejiang, China.
PLoS One. 2018 Oct 8;13(10):e0205182. doi: 10.1371/journal.pone.0205182. eCollection 2018.
Empoasca onukii Matsuda is one of the most devastating pests of the tea plant (Camellia sinensis). Still, the presumed expression stability of its reference genes (RGs) has not been analyzed. RGs are essential for accurate and reliable gene expression analysis, so this absence has hampered the study of the insect's molecular biology. To find candidate RGs for normalizing gene expression data, we cloned ten common housekeeping genes from E. onukii. Using the ΔCt method, geNorm, NormFinder and BestKeeper, we screened the RGs that were appropriate for quantifying the mRNA transcription of cellular responses under five experimental conditions. We identified the combinations of α-TUB and G6PDH, α-TUB and UBC, two RGs (α-TUB and β-TUB1) or three RGs (α-TUB, RPL13 and GAPDH), AK and UBC, or RPL13 and α-TUB as the best for analyzing gene expression in E. onukii adults of both sexes in different tissues, nymphs at different developmental stages, nymphs exposed to different temperatures or nymphs exposed to photoperiod stress. Finally, the E. onukii cysteine proteinase (Eocyp) was chosen as the target gene to validate the rationality of the proposed RGs. In conclusion, our study suggests a series of RGs with which to study the gene expression profiles of E. onukii that have been manipulated (biotically or abiotically) using reverse transcription quantitative polymerase chain reaction. The results offer a solid foundation for further studies of the molecular biology of E. onukii.
茶黄蓟马（Empoasca onukii Matsuda）是茶树（Camellia sinensis）上最具破坏性的害虫之一。然而，其假定的参考基因（RGs）表达稳定性尚未被分析。RGs 是准确可靠的基因表达分析的基础，因此这种缺失阻碍了昆虫分子生物学的研究。为了找到用于归一化基因表达数据的候选 RGs，我们从 E. onukii 克隆了十个常见的管家基因。使用 ΔCt 方法、geNorm、NormFinder 和 BestKeeper，我们筛选了适合在五种实验条件下量化细胞反应的 mRNA 转录的 RGs。我们确定了 α-TUB 和 G6PDH、α-TUB 和 UBC、两个 RG（α-TUB 和 β-TUB1）或三个 RG（α-TUB、RPL13 和 GAPDH）、AK 和 UBC 或 RPL13 和 α-TUB 作为分析两性成虫在不同组织、不同发育阶段的若虫、暴露于不同温度的若虫或暴露于光周期胁迫的若虫中基因表达的最佳组合。最后，选择茶黄蓟马半胱氨酸蛋白酶（Eocyp）作为靶基因，验证所提出的 RG 的合理性。总之，我们的研究提出了一系列 RGs，可用于研究使用反转录定量聚合酶链反应（RT-qPCR）处理过（生物或非生物）的 E. onukii 的基因表达谱。这些结果为进一步研究 E. onukii 的分子生物学提供了坚实的基础。
