Comparison of the MpEF1α and CaMV35 promoters for application in Marchantia polymorpha overexpression studies
Comparison of the MpEF1α and CaMV35 promoters for application in Marchantia polymorpha overexpression studies
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DOI：
10.1007/s11248-013-9746-z
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发表时间：
2014-04-01
影响因子：
3
通讯作者：
Zachgo, Sabine
中科院分区：
文献类型：
--
作者：
Althoff, Felix;Kopischke, Sarah;Zachgo, Sabine
组成型启动子是利用转基因手段分析基因功能的重要工具。对于基因的过度表达和沉默研究，普遍存在的和强表达的被研究基因以及选择标记是首选。分析了病毒35S花椰菜花叶病毒启动子和内源延伸因子1α(MpEF1α)启动子的活性，为其在新的基础植物模式系统中的应用奠定了基础。报告基因β-葡萄糖醛酸苷酶(GUS)在CaMV35和MpEF1α启动子的驱动下，在整个植物发育过程中的表达进行了比较。两种启动子的活性有显著差异。CaMV35启动子在分生组织区产生弱的报告基因表达，但在菌体中强表达。MpEF1α启动子引起强烈的分生组织GUS表达，并且在女性的性组织中更活跃。总体而言，MpEF1α启动子似乎是获得强大且普遍存在的转基因表达的更好选择。此外，还建立了一套完整的地钱草原位杂交方法。对完整的整个组织中MpEF1αmRNA转录本的分析显示，表达模式总体上与MpEF1α启动子驱动的GUS报告基因的表达模式相似，包括在分生区的强表达。该技术可用于检测完整的生殖器和颈卵器中的mRNA表达，并有可能应用于大量转基因植物的筛选，例如鉴定击倒突变体。
Constitutive promoters are essential tools for analyses of gene functions by transgenic approaches. For overexpression and silencing studies of genes, a ubiquitous and strong expression of genes under investigation as well as selection markers is preferred. For future applications in the emerging basal plant model system Marchantia polymorpha, a liverwort, activities of the viral 35S cauliflower mosaic virus promoter and the endogenous elongation factor 1 alpha (MpEF1 alpha) promoter were analyzed. Expression of the reporter gene beta-glucuronidase (GUS), driven by the CaMV35 and MpEF1 alpha promoters, was compared throughout plant development. Significant differences were observed between the two promoter activities. The CaMV35 promoter yields a weak reporter gene expression in the meristematic zones but drives a strong expression in the thallus. The MpEF1 alpha promoter causes a strong meristematic GUS expression and is more active in female sexual tissues. Overall, the MpEF1 alpha promoter seems to be the better option for obtaining a strong and ubiquitous transgene expression. Furthermore, a whole mount in situ hybridization protocol for Marchantia was established. Analysis of MpEF1 alpha mRNA transcript in intact, whole tissues showed an expression pattern that is overall similar to the pattern of the GUS reporter gene expression driven by the MpEF1 alpha promoter, including strong expression in meristematic zones. The whole mount technique reported here can be used to determine the mRNA expression in intact gemmae and archegonia, and has the potential to be applied for screening large numbers of transgenic plants, for instance to identify knock-down mutants.