title: Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) Using real-time PCR.
abstract: Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR). In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG), and abscisic acid (ABA). Three analytical software packages (geNorm, Bestkeeper, and NormFinder) were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC) and alpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, People's Republic of China.
authors.forename: Ermei
authors.initials: E
authors.lastname: Chang
authors.forename: Shengqing
authors.initials: S
authors.lastname: Shi
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Liu
authors.forename: Tielong
authors.initials: T
authors.lastname: Cheng
authors.forename: Liang
authors.initials: L
authors.lastname: Xue
authors.forename: Xiuyan
authors.initials: X
authors.lastname: Yang
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Yang
authors.forename: Qian
authors.initials: Q
authors.lastname: Lan
authors.forename: Zeping
authors.initials: Z
authors.lastname: Jiang
mesh_terms: D000040:Abscisic Acid / Q000494:pharmacology; D001483:Base Sequence; D003080:Cold Temperature; D029779:Cupressaceae / Q000235:genetics* / Q000254:growth & development; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010937:Plant Growth Regulators / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors
chemical_list: D010937:Plant Growth Regulators; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D000040:Abscisic Acid