Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Normalization in Staminate and Perfect Flowers of Andromonoecious Taihangia rupestris.
Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Normalization in Staminate and Perfect Flowers of Andromonoecious Taihangia rupestris.
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选择并验证雄花同株太行花雄花和完美花实时定量 PCR 标准化的内参基因
DOI：
10.3389/fpls.2017.00729
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发表时间：
2017
影响因子：
5.6
通讯作者：
Zhang Y
中科院分区：
文献类型：
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作者：
Li W;Zhang L;Zhang Y;Wang G;Song D;Zhang Y
定量实时逆转录聚合酶链反应（qRT-PCR）是基因表达分析中最常用和最强大的方法，因为它具有高灵敏度、特异性和高通量，而这种方法的准确性取决于用于归一化的内参基因的稳定性。太行花玉李（蔷薇科）是一种雄花雌雄同株的植物，在同一个体内产生两性花和单性雄花。利用qRT-PCR技术研究雄蕊花和完成花的基因表达谱，有助于进一步了解雄蕊花形成和性别分化调控的分子机制。为了准确规范太行花基因表达水平，基于转录组序列数据和已有研究，选择了16个候选内参基因，其中包括10个传统内参基因和6个新稳定基因。采用qRT-PCR分析了51个样品中这些基因的表达，其中包括30个不同发育阶段的雄花和完花样品以及21个成熟花的不同花组织样品。通过geNorm、NormFinder、BestKeeper和综合RefFinder算法，确定ADF3与UFD1组合为雄花的最佳内参基因，而HIS3/ADF3组合为完美花样品最准确的内参基因。对于花组织，HIS3、UFD1和TMP50是最合适的内参基因。此外，我们选择了两个参与花器官鉴定的靶基因TruPI和TruFBP24，验证了雄蕊花、完美花和不同花组织中最稳定和最不稳定的内参基因，表明使用不合适的内参基因进行规范化将导致不良结果。本研究鉴定的内参基因将提高雄雄雌雄同株红桃花中目的基因表达qRT-PCR定量的准确性，为今后花发育和性别分化的功能基因组学研究奠定基础。
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used and powerful method for gene expression analysis due to its high sensitivity, specificity, and high throughput, and the accuracy of this approach depends on the stability of reference genes used for normalization. Taihangia rupestris Yu and Li (Rosaceae), an andromonoecious plant, produces both bisexual flowers and unisexual male flowers within the same individual. Using qRT-PCR technique, investigation of the gene expression profiling in staminate and perfect flowers would improve our understanding of the molecular mechanism in regulation of flower formation and sex differentiation in andromonoecious T. rupestris. To accurate normalize the gene expression level in Taihangia flower, 16 candidate reference genes, including 10 traditional housekeeping genes, and 6 newly stable genes, were selected based on transcriptome sequence data and previous studies. The expressions of these genes were assessed by qRT-PCR analysis in 51 samples, including 30 staminate and perfect flower samples across developmental stages and 21 different floral tissue samples from mature flowers. By using geNorm, NormFinder, BestKeeper, and comprehensive RefFinder algorithms, ADF3 combined with UFD1 were identified as the optimal reference genes for staminate flowers, while the combination of HIS3/ADF3 was the most accurate reference genes for perfect floral samples. For floral tissues, HIS3, UFD1, and TMP50 were the most suitable reference genes. Furthermore, two target genes, TruPI, and TruFBP24, involved in floral organ identity were selected to validate the most and least stable reference genes in staminate flowers, perfect flowers, and different floral tissues, indicating that the use of inappropriate reference genes for normalization will lead to the adverse results. The reference genes identified in this study will improve the accuracy of qRT-PCR quantification of target gene expression in andromonoecious T. rupestris flowers, and will facilitate the functional genomics studies on flower development and sex differentiation in the future.