Timm Stefan, Modde Katharina, Bauwe Hermann
Plant Physiology Department, University of Rostock, Albert-Einstein-Straße 3, 18059, Rostock, Germany.
Methods Mol Biol. 2017;1653:105-124. doi: 10.1007/978-1-4939-7225-8_8.
Transfer DNA (T-DNA) insertional lines have facilitated reverse genetic approaches in plant science and considerably accelerated the functional characterization of genes. Typically, online databases of mutant collections are searched for suitable mutant alleles of the gene of interest (GOI). Before such lines can be characterized physiologically, the genotype of the respective mutant has to be verified followed by the quantitative examination of downstream effects on the levels of the respective mRNA and the encoded protein. Here, we describe a typical workflow for the identification of photorespiratory mutants followed by phenotypic characterization according to growth under different conditions, photosynthesis on the levels of chlorophyll a fluorescence and gas exchange, and metabolite analysis.
转移DNA（T-DNA）插入系促进了植物科学中的反向遗传学方法，并大大加快了基因的功能表征。通常，会在突变体文库的在线数据库中搜索感兴趣基因（GOI）的合适突变等位基因。在对这些品系进行生理特征分析之前，必须先验证各个突变体的基因型，然后定量检测其对相应mRNA和编码蛋白水平的下游影响。在这里，我们描述了一种典型的工作流程，用于鉴定光呼吸突变体，然后根据不同条件下的生长情况、叶绿素a荧光水平和气体交换方面的光合作用以及代谢物分析进行表型特征分析。
