Evaluation of Reference Genes for RT qPCR Analyses of Structure-Specific and Hormone Regulated Gene Expression in Physcomitrella patens Gametophytes

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions.小立碗藓（Physcomitrella patens）作为研究植物发育与生理学的模式系统，其应用正快速扩张。该物种在绿色植物演化谱系中处于藻类与维管植物之间的关键位置，同源重组介导的转基因整合效率极高，其单倍体配子体作为生活史的主导阶段具有独特优势，构成单倍体配子体的原丝体、叶状枝与假根结构简单，加之其高质量基因组序列易于获取，这些特质使该苔藓成为极具吸引力的实验体系。针对小立碗藓发育过程的遗传与激素调控研究，高度依赖通过实时定量聚合酶链式反应（real time quantitative PCR，RT-qPCR）分析基因表达模式。该技术需要经过充分表征的内参基因集——这类基因在所有分析条件下的表达水平变异极小——用于数据归一化处理。目前多数主流模式系统均已报道了适配的内参基因集，例如拟南芥（Arabidopsis thaliana），但针对小立碗藓的相关研究仍为空白。本研究基于RT-qPCR技术，对12个候选内参基因的转录水平，在一系列不同发育阶段的小立碗藓配子体结构，以及经激素或激素运输抑制剂处理的小立碗藓原丝体中开展了比较分析。通过GeNorm与NormFinder软件对上述RT-qPCR数据进行分析，本研究成功鉴定出适用于所有测试条件下基因表达分析的小立碗藓内参基因集，并发现各类测试条件下仅需选取2个最优内参基因即可实现有效的数据归一化处理。