Evaluation of reference genes for RT qPCR analyses of structure-specific and hormone regulated gene expression in Physcomitrella patens gametophytes.
Evaluation of reference genes for RT qPCR analyses of structure-specific and hormone regulated gene expression in Physcomitrella patens gametophytes.
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DOI：
10.1371/journal.pone.0070998
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Kost B
中科院分区：
文献类型：
--
作者：
Le Bail A;Scholz S;Kost B
利用小壶藓（Physcomitrella patens）作为研究植物发育和生理的模式系统正在迅速扩大。在藻类和维管植物之间的绿色系谱中的战略地位，同源重组对转基因的高效整合，单倍体配子体的优势，单倍体配子体的简单结构，构成单倍体配子体的叶状芽和根状体，以及易于获得的高质量基因组序列使这种苔藓成为一个非常有吸引力的实验系统。实时定量PCR （real - time quantitative PCR, RT - qPCR）技术在很大程度上依赖于基因表达模式的分析。该技术需要具有良好特征的内参基因集，在所有分析条件下显示最小的表达水平变化，以便进行数据规范化。对拟南芥等最广泛使用的模式系统，已经描述了合适的内参基因，但对拟南芥则没有。在此，我们采用RT - qPCR方法比较了不同发育阶段的patens的配子体结构和激素或激素运输抑制剂处理的patens原体中12个候选内参基因的转录水平。利用GeNorm和NormFinder软件对这些RT - qPCR数据进行分析，鉴定出了在所有测试条件下适合基因表达分析的一组专利草内参基因，并表明两个最佳内参基因足以在每种条件下进行有效的数据归一化。
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.
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