Moebes Michael, Kuhlmann Heike, Demidov Dmitri, Lermontova Inna
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany.
Front Plant Sci. 2022 Aug 9;13:954976. doi: 10.3389/fpls.2022.954976. eCollection 2022.
Rapeseed () is an allopolyploid hybrid (AACC genome) of turnip rape (, genome: AA) and vegetable cabbage (, genome: CC). Rapeseed oil is one of the main vegetable oils used worldwide for food and other technical purposes. Therefore, breeding companies worldwide are interested in developing rapeseed varieties with high yields and increased adaptation to harsh climatic conditions such as heat and prolonged drought. One approach to studying the mechanism of the epigenetically regulated stress response is to analyze the transcriptional changes it causes. In addition, comparing the expression of certain genes between stress- and non-stress-tolerant varieties will help guide breeding in the desired direction. Quantitative reverse transcription PCR (RT-qPCR) has been intensively used for gene expression analysis for several decades. However, the transfer of this method from model plants to crop species has several limitations due to the high accumulation of secondary metabolites, the higher water content in some tissues and therefore problems with their grinding and other factors. For allopolyploid rapeseed, the presence of two genomes, often with different levels of expression of homeologous genes, must also be considered. In this study, we describe the optimization of transcriptional RT-qPCR analysis of low-expression epigenetic genes in rapeseed, using (), a regulator of kinetochore complex assembly, as an example. We demonstrated that a combination of various factors, such as tissue homogenization and RNA extraction with TRIzol, synthesis of cDNA with gene-specific primers, and RT-qPCR in white plates, significantly increased the sensitivity of RT-qPCR for the detection of and gene expression.
油菜（）是芜菁油菜（，基因组：AA）和青菜（，基因组：CC）的异源多倍体杂交种（AACC基因组）。菜籽油是全球用于食品和其他技术用途的主要植物油之一。因此，全球的育种公司都对培育高产且能更好适应高温和长期干旱等恶劣气候条件的油菜品种感兴趣。研究表观遗传调控应激反应机制的一种方法是分析其引起的转录变化。此外，比较抗逆和不抗逆品种之间某些基因的表达将有助于朝着期望的方向指导育种。几十年来，定量逆转录PCR（RT-qPCR）一直被广泛用于基因表达分析。然而，由于次生代谢产物的大量积累、某些组织中较高的含水量以及由此带来的研磨问题等其他因素，该方法从模式植物转移到作物物种存在一些局限性。对于异源多倍体油菜，还必须考虑两个基因组的存在，它们的同源基因表达水平往往不同。在本研究中，我们以动粒复合体组装的调节因子（）为例，描述了油菜中低表达表观遗传基因转录RT-qPCR分析的优化。我们证明，多种因素的组合，如用TRIzol进行组织匀浆和RNA提取、用基因特异性引物合成cDNA以及在白色板中进行RT-qPCR，显著提高了RT-qPCR检测和基因表达的灵敏度。
