College of Horticulture, Henan Agricultural University, 63 Nongye Road, Zhengzhou, 450002, China.
College of Forestry, Henan Agricultural University, 63 Nongye Road, Zhengzhou, 450002, China.
Mol Biol Rep. 2019 Dec;46(6):6027-6037. doi: 10.1007/s11033-019-05038-5. Epub 2019 Aug 30.
Begonia semperflorens (B. semperflorens), belonging to the family Begoniaceae, has now been widely cultivated worldwide and is famous for its ornamental plants with colourful flowers and distinctive leaves. The selection of appropriate internal reference genes is very important to accurately determine target gene expression via quantitative real-time PCR. However, internal reference gene selection has never been conducted in B. semperflorens. In this study, seven candidate reference genes of B. semperflorens, including 18S ribosomal RNA (Bs18S), pentatricopeptide repeat-containing protein (BsPPR), actin-related protein 5 isoform X2 (BsACT), DNAJ homologue subfamily C member 17 (BsDNAJ), glyceraldehyde-3-phosphate dehydrogenase (BsGAPDH), NAD-dependent malic enzyme 59 kDa isoform, mitochondria (BsNAD-ME), and peptidyl-prolyl cis-trans isomerase CYP26-2, chloroplast (BsCYP), which were obtained from our previous studies, were selected. The stabilities of these genes under stress conditions were analysed using geNorm and NormFinder. Validation of target gene expressions, including phenylalanine ammonia-lyase (BsPAL) and respiratory burst oxidase homologue D (BsRBOHD) under biotic and abiotic conditions, phenylalanine ammonia-lyase (BsPAL), anthocyanidin synthase (BsANS), chalcone synthase (BsCHS), and flavanone-3-hydroxylase (BsF3H) under low temperature, using these seven internal reference genes for normalisation further confirmed the stabilities of the selected genes and indicated the need for reference gene selection for normalising gene expressions in B. semperflorens. Of the seven candidate reference genes, the combination of BsACT, BsDNAJ, and BsNAD-ME was the ideal reference gene set for normalising gene expression in samples under biotic conditions. BsCYP combined with BsACT or BsGAPDH was the best reference gene pair under abiotic conditions. BsACT and BsPPR could be combined to normalise gene expression under low temperature. Our results will benefit future studies on gene expression in plants of Begoniaceae.
四季秋海棠（B. semperflorens），属于秋海棠科，现已在全球范围内广泛种植，以其色彩斑斓的花朵和独特的叶子而闻名的观赏植物。通过定量实时 PCR 准确确定靶基因表达，选择合适的内参基因非常重要。然而，在四季秋海棠中从未进行过内参基因的选择。在这项研究中，从我们之前的研究中选择了 7 个四季秋海棠候选内参基因，包括 18S 核糖体 RNA（Bs18S）、五肽重复蛋白（BsPPR）、肌动蛋白相关蛋白 5 同工型 X2（BsACT）、DNAJ 同源物亚家族 C 成员 17（BsDNAJ）、甘油醛-3-磷酸脱氢酶（BsGAPDH）、NAD-依赖性苹果酸酶 59 kDa 同工型、线粒体（BsNAD-ME）和肽基脯氨酰顺反异构酶 CYP26-2、叶绿体（BsCYP）。使用 geNorm 和 NormFinder 分析这些基因在应激条件下的稳定性。验证靶基因表达，包括生物和非生物条件下苯丙氨酸解氨酶（BsPAL）和呼吸爆发氧化酶同源物 D（BsRBOHD）、生物条件下苯丙氨酸解氨酶（BsPAL）、花青素合酶（BsANS）、查尔酮合酶（BsCHS）和黄烷酮-3-羟化酶（BsF3H）在低温下使用这 7 个内参基因进行归一化进一步证实了所选基因的稳定性，并表明需要选择内参基因来归一化四季秋海棠中的基因表达。在 7 个候选内参基因中，BsACT、BsDNAJ 和 BsNAD-ME 的组合是生物条件下基因表达归一化的理想内参基因集。非生物条件下，BsCYP 与 BsACT 或 BsGAPDH 结合是最佳的参考基因对。BsACT 和 BsPPR 可以结合起来在低温下归一化基因表达。我们的研究结果将有助于今后对秋海棠科植物基因表达的研究。
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