Dzialo Magdalena, Szopa Jan, Czuj Tadeusz, Zuk Magdalena
Department of Genetic Biochemistry, Faculty of Biotechnology, University of WrocławWroclaw, Poland.
Linum FoundationWroclaw, Poland.
Front Plant Sci. 2017 May 15;8:755. doi: 10.3389/fpls.2017.00755. eCollection 2017.
Chalcone synthase (CHS) has been recognized as an essential enzyme in the phenylpropanoid biosynthesis pathway. Apart from the leading role in the production of phenolic compounds with many valuable biological activities beneficial to biomedicine, CHS is well appreciated in science. Genetic engineering greatly facilitates expanding knowledge on the function and genetics of CHS in plants. The CHS gene is one of the most intensively studied genes in flax. In our study, we investigated engineering of the CHS gene through genetic and epigenetic approaches. Considering the numerous restrictions concerning the application of genetically modified (GM) crops, the main purpose of this research was optimization of the plant's modulation via epigenetics. In our study, plants modified through two methods were compared: a widely popular agrotransformation and a relatively recent oligodeoxynucleotide (ODN) strategy. It was recently highlighted that the ODN technique can be a rapid and time-serving antecedent in quick analysis of gene function before taking vector-mediated transformation. In order to understand the molecular background of epigenetic variation in more detail and evaluate the use of ODNs as a tool for predictable and stable gene engineering, we concentrated on the integration of gene expression and gene-body methylation. The treatment of flax with a series of short oligonucleotides homologous to a different part of CHS gene isoforms revealed that those directed to regulatory gene regions (5'- and 3'-UTR) activated gene expression, directed to non-coding region (introns) caused gen activity reduction, while those homologous to a coding region may have a variable influence on its activity. Gene expression changes were accompanied by changes in its methylation status. However, only certain (CCGG) motifs along the gene sequence were affected. The analyzed DNA motifs of the CHS flax gene are more accessible for methylation when located within a CpG island. The methylation motifs also led to rearrangement of the nucleosome location. The obtained results suggest high specificity of ODN action and establish a potential valuable alternative for improvement of crops.
查尔酮合酶（CHS）被认为是苯丙烷生物合成途径中的一种关键酶。除了在生产具有许多对生物医学有益的重要生物活性的酚类化合物中起主导作用外，CHS在科学界也备受赞誉。基因工程极大地促进了对植物中CHS功能和遗传学的认识扩展。CHS基因是亚麻中研究最为深入的基因之一。在我们的研究中，我们通过遗传和表观遗传方法研究了CHS基因工程。考虑到转基因作物应用存在的诸多限制，本研究的主要目的是通过表观遗传学优化植物调控。在我们的研究中，比较了通过两种方法改造的植物：一种是广泛应用的农杆菌介导转化法，另一种是相对较新的寡脱氧核苷酸（ODN）策略。最近有研究强调，在进行载体介导的转化之前，ODN技术可以作为一种快速且省时的方法，用于基因功能的快速分析。为了更详细地了解表观遗传变异的分子背景，并评估ODN作为一种可预测和稳定基因工程工具的用途，我们专注于基因表达与基因体甲基化的整合。用一系列与CHS基因异构体不同部分同源的短寡核苷酸处理亚麻后发现，那些靶向调控基因区域（5'-和3'-UTR）的寡核苷酸激活了基因表达，靶向非编码区域（内含子）的寡核苷酸导致基因活性降低，而那些与编码区域同源的寡核苷酸可能对其活性有不同影响。基因表达变化伴随着其甲基化状态的改变。然而，只有基因序列上特定的（CCGG）基序受到影响。当位于CpG岛内时，亚麻CHS基因的分析DNA基序更容易发生甲基化。甲基化基序还导致核小体位置的重排。所得结果表明ODN作用具有高度特异性，并为作物改良建立了一种潜在的有价值的替代方法。
