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首页> 外文期刊>Journal of Experimental Marine Biology and Ecology >Selection and validation of reference genes for qPCR analysis in the pennate diatoms Pseudo-nitzschia multistriata and P. arenysensis
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Selection and validation of reference genes for qPCR analysis in the pennate diatoms Pseudo-nitzschia multistriata and P. arenysensis

机译：羽状硅藻（Pseudo-Nitzschia multistriata）和槟榔（P. arenysensis）的qPCR分析参考基因的选择和验证

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摘要

Diatoms are eukaryotic microalgae broadly present in freshwater and marine ecosystems. The great ecological interest about diatoms has recently led to increased efforts towards the exploration of the molecular properties of these organisms and the development of molecular tools to study the function of genes. We are using as model system two pennate diatoms, Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis. In order to enable molecular investigations in these two species, we have made use of sequence information from transcriptomic data to identify genes that can be used as reference genes in quantitative PCR (qPCR). We have analyzed the expression of a set of genes commonly used as reference genes: histone H4, TBP (TATA binding protein), RPS (30 S ribosomal protein), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), TUB A and TUB B (tubulin α and tubulin β), ACT (actin), CDK A (cyclin dependent kinase A) and COPA (coatomer protein complex, subunit α). The suitability of these genes as references in qPCR has been tested in different conditions and among different strains. We have found that only TUB A, TUB B and CDKA are stable in all the conditions analyzed. These three genes, in addition to ACT and COPA, are good reference genes for P. multistriata, while CAPDH appears to be differentially expressed in the tested conditions in this species. In P. arenysensis, instead, TUB A, TUB B, CDK A, GAPDH, H4 and RPS show the highest levels of stability and can be considered reliable reference genes.
机译：硅藻是真核微藻，广泛存在于淡水和海洋生态系统中。关于硅藻的巨大生态兴趣最近导致人们加大了对这些生物的分子特性的探索，并为研究基因功能开发了分子工具。我们正在使用两个假的硅藻作为模型系统，即多假拟南芥和斑假拟南芥。为了能够对这两个物种进行分子研究，我们利用了转录组数据中的序列信息来鉴定可用作定量PCR（qPCR）参考基因的基因。我们分析了一组通常用作参考基因的基因的表达：组蛋白H4，TBP（TATA结合蛋白），RPS（30 S核糖体蛋白），GAPDH（3-磷酸甘油醛脱氢酶），TUB A和TUB B（微管蛋白） α和微管蛋白β），ACT（肌动蛋白），CDK A（细胞周期蛋白依赖性激酶A）和COPA（涂层蛋白复合物，α亚基）。这些基因在qPCR中作为参考的适用性已在不同条件下和不同菌株之间进行了测试。我们发现，在所有分析的条件下，只有TUB A，TUB B和CDKA是稳定的。除ACT和COPA外，这三个基因是多纹假单胞菌的良好参考基因，而CAPDH在该物种的测试条件下似乎差异表达。相反，在槟榔对虾中，TUB A，TUB B，CDK A，GAPDH，H4和RPS显示出最高的稳定性水平，可以认为是可靠的参考基因。

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