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Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization

机译:通过选择均一转录的mRNA作为归一化的基础来量化海中硅藻基因的表达

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To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, TBP (encoding the TATA box-binding protein) and EFL (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of TBP and EFL were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in Skeletonema costatum and Chaetoceros affinis , and TBP expression was more stable than that of EFL . Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 EFL and 29 TBP homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, EFL and TBP primer sets were designed for Chaetoceros and Skeletonema groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the EFL primer sets performed better. To demonstrate the applicability of EFL primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in FcpB expression and confirmed EFL as a good reference gene.
机译:为了通过天然硅藻组合物中的定量逆转录PCR(Q-RT-PCR)定量基因表达,有必要寻找特定于目标物种的生物量参考。在Q-RT-PCR分析中,评估了两个管家基因TBP(编码TATA盒结合蛋白)和EFL(编码翻译延伸因子样蛋白)作为参考基因的候选基因。 TBP和EFL的转录本水平在各种测试条件下都相对稳定,包括生长阶段,明暗循环阶段以及肋骨和Chaetoceros亲缘关系中的营养胁迫,并且TBP表达比EFL更稳定。接下来,通过从东海(ECS)获得32个EFL和29个TBP同源基因片段来评估硅藻组装体的序列多样性。根据序列比对,为ECS中的Chaetoceros和Skeletonema组设计了EFL和TBP引物组。对引物特异性和PCR效率的评估表明,EFL引物组表现更好。为了证明EFL引物组在ECS中的适用性,使用它们来测量硅藻中FcpB(岩藻黄质-叶绿素蛋白)基因的mRNA水平。结果正确地揭示了FcpB表达中显着的diel变异,并证实EFL是良好的参考基因。

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