麦长管蚜miRNA实时荧光定量PCR内参基因的筛选

【目的】筛选特定条件下麦长管蚜Sitobion avenae稳定表达的微小RNA(miRNA)表达分析内参基因。【方法】根据麦长管蚜miRNA Illumina测序结果筛选出miR-10-3p, miR-993, miR-276, miR-275, miR-252a, miR-1, miR-375, pc-15, pc-73和1个常用内参基因U6共10个候选内参基因。利用qRT-PCR测定10个候选内参基因在麦长管蚜有翅蚜和无翅蚜不同发育时期、不同组织以及4种化学药剂(95.1%吡虫啉原药、97.8%噻虫啉原药、95%阿维菌素原药和40%氧乐果乳油)处理无翅蚜中的相对表达量。利用GeNorm, NormFinder, ΔCt法, BestKeeper和RefFinder对10个候选内参基因的表达稳定性进行评价。【结果】qRT-PCR结果表明，在麦长管蚜有翅蚜不同发育阶段、无翅蚜不同发育阶段、有翅蚜和无翅蚜不同组织中和4种药剂处理的无翅蚜中表达量较高的内参基因分别是miR-276, miR-276, miR-10-3p和miR-10-3p；5种方法综合分析得出，在上述4种条件下表达稳定性都较好的内参基因分别是miR-252a, miR-993, miR-275和miR-993。GeNorm软件分析表明不同条件下麦长管蚜内参基因的较佳数目为2，结合RefFinder稳定性综合排序的结果分析可知在有翅蚜不同发育阶段、无翅蚜不同发育阶段、有翅蚜和无翅蚜不同组织中和不同药剂处理无翅蚜中稳定表达的内参基因组合分别是miR-252a和miR-276, miR-993和miR-276, miR-275和miR-10-3p, miR-993和miR-10-3p。【结论】麦长管蚜有翅蚜不同发育阶段、无翅蚜不同发育阶段、有翅蚜和无翅蚜不同组织中和不同药剂处理无翅蚜中内参基因优化组合分别为miR-252a和miR-276, miR-993和miR-276, miR-275和miR-10-3p, miR-993和miR-10-3p。研究结果为麦长管蚜miRNA基因定量表达的研究提供了内参基因选择的依据。

Selection of reference genes for real-time fluorescence quantitative PCR of miRNAs in Sitobion avenae (Hemiptera: Aphididae)

【Aim】 This study aims to select the stably expressed reference genes for expression analysis of microRNAs (miRNAs) in the cereal aphid, Sitobion avenae, under specific conditions. 【Methods】 According to the Illumina sequencing results of S. avenae miRNAs, 10 candidate reference genes including miR-10-3p, miR-993, miR-276, miR-275, miR-252a, miR-1, miR-375, pc-15, pc-73 and one normal reference gene U6 were screened, and their relative expression levels in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids and in wingless aphids of S. avenae exposed to four insecticides (95.1% imidacloprid technical material, 97.8% thiacloprid technical material, 95% avermectins technical material and 40% omethoate emulsifiable concentrate) were detected by using qRT-PCR. The expression stability of the 10 candidate reference genes was evaluated by GeNorm, NormFinder, ΔCt method, BestKeeper, and RefFinder. 【Results】 The qRT-PCR results showed that the highly expressed reference genes in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids, and in wingless aphids of S. avenae exposed to four insecticides were miR-276, miR-276, miR-10-3p, and miR-10-3p, respectively. Integrating the analysis results of five approaches, more stably expressed reference genes under the above four conditions were miR-252a, miR-993, miR-275, and miR-993, respectively. The optimal number of reference genes was two under different conditions by GeNorm. Based on the comprehensive ranking of RefFinder, the optimal combinations of stably expressed reference genes in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids, and in wingless aphids of S. avenae exposed to four insecticides were miR-252a and miR-276, miR-993 and miR-276, miR-275 and miR-10-3p, miR-993 and miR-10-3p, respectively. 【Conclusion】 The optimal combinations of reference genes in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids, and in wingless aphids of S. avenae exposed to different insecticides are miR-252a and miR-276, miR-993 and miR-276, miR-275 and miR-10-3p, miR-993 and miR-10-3p, respectively. The results provide a basis of reference gene selection for quantitative expression analysis of miRNA genes in S. avenae.