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首页> 外文期刊>BMC Plant Biology >Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha
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Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha

机译：无融合生殖和有性草臂形臂锈菌中用于定量实时PCR表达研究的参考基因的选择

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Background Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur. Results Eight genes were selected from a cDNA library of ovaries of B. brizantha considering the similarity to reference genes: EF1 (elongation factor 1 alpha), E1F4A (eukaryotic initiation factor 4A), GAPDH (glucose-6-phosphate dehydrogenase), GDP (glyceroldehyde-3-phosphate dehydrogenase), SUCOA (succinyl-CoA ligase), TUB (tubulin), UBCE (ubiquitin conjugating enzyme), UBI (ubiquitin). For the analysis, total RNA was extracted from 22 samples and raw Ct data after qRT-PCR reaction was analyzed for primer efficiency and for an overall analysis of Ct range among the different samples. Elongation factor 1 alpha showed the highest expression levels, whereas succinyl-CoA ligase showed the lowest within the chosen set of samples. GeNorm application was used for evaluation of the best reference genes, and according to that, the least stable genes, with the highest M values were tubulin and succinyl-CoA ligase and the most stable ones, with the lowest M values were elongation factor 1 alpha and ubiquitin conjugating enzyme, when both reproductive and vegetative samples were tested. For ovaries and spikelets of both sexual and apomictic B. brizantha the genes with the lowest M values were Bbriz UBCE, Bbriz E1F4A and Bbriz EF1. Conclusion In total, eight genes belonging to different cellular processes were tested. Out of them, Bbriz TUB was the less stable while Bbriz EF1 followed by Bbriz UBCE were the more stable genes considering male and female reproductive tissues, spikelets, roots and leaves. Regarding the best reference genes for ovary tissues, where apomictic and sexual reproduction must occur, the best reference genes were Bbriz UBCE, Bbriz E1F4A and Bbriz EF1. Our results provide crucial information for transcriptional analysis in the Brachiaria ssp, helping to improve the quality of gene expression data in these species, which constitute an excellent plant system for the study of apomixis.
机译：背景Brachiaria brizantha是一种重要的牧草。腕足动物中无融合生殖和有性生殖的发生，使其成为理解两种生殖方式涉及的分子途径的有趣系统。定量实时PCR（qRT-PCR）已经成为一种比较目标基因表达谱的重要技术，为了获得可靠的结果，拥有合适的参考基因非常重要。在这项工作中，我们评估了B. brizantha qRT-PCR实验的八个潜在参考基因，这些参考基因是从cDNA卵巢文库中分离的。测试了无融合生殖和有性B. brizantha的营养和生殖组织，以验证参考基因，包括雌配子体，其中有性和无融合生殖植物之间必须发生表达差异。结果从B. brizantha卵巢的cDNA文库中选择了八个基因，考虑到它们与参考基因的相似性：EF1（延伸因子1α），E1F4A（真核起始因子4A），GAPDH（葡萄糖6-磷酸脱氢酶），GDP（甘油三磷酸脱氢酶），SUCOA（琥珀酰辅酶A连接酶），TUB（微管蛋白），UBCE（泛素结合酶），UBI（泛素）。为了进行分析，从22个样品中提取了总RNA，并对qRT-PCR反应后的原始Ct数据进行了引物效率分析和不同样品之间Ct范围的整体分析。延伸因子1α显示最高的表达水平，而琥珀酰辅酶A连接酶显示在所选的样品组中最低。 GeNorm应用程序用于评估最佳参考基因，因此，M值最高的最不稳定基因是微管蛋白和琥珀酰-CoA连接酶，M值最低的最稳定基因是延伸因子1 alpha。同时检测生殖和营养样品时的泛素和泛素结合酶。对于性和无融合性B. brizantha的卵巢和小穗，具有最低M值的基因是Bbriz UBCE，Bbriz E1F4A和Bbriz EF1。结论总共测试了八个属于不同细胞过程的基因。其中，考虑到雄性和雌性生殖组织，小穗，根和叶，Bbriz TUB的稳定性较差，而Bbriz EF1其次是Bbriz UBCE则更稳定。关于必须发生无融合生殖和有性生殖的卵巢组织的最佳参考基因，最佳参考基因是Bbriz UBCE，Bbriz E1F4A和Bbriz EF1。我们的结果为腕带属菌种的转录分析提供了关键信息，有助于提高这些物种中基因表达数据的质量，从而构成了研究无融合生殖的优良植物系统。

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