紫玉兰‘红元宝'花芽分化阶段基因定量分析的内参基因筛选

Selection of reference genes in Magnolia liliflora ‘Hongyuanbao' during flower bud differentiation

关键词:  紫玉兰, 内参基因, 两次成花, qRT-PCR, 微管蛋白基因

Key words:  Magnolia liliflora, reference gene, re-flowering, qRT-PCR, TUB

摘要:

为筛选紫玉兰‘红元宝'(Magnolia liliflora ‘Hongyuanbao')二次花芽分化阶段稳定表达的内参基因,该研究以‘红元宝'不同花芽分化时期的花芽和叶为材料,基于转录组数据,筛选出8个候选内参基因,即泛素酶基因(UBC)、肌动蛋白(ACT)、微管蛋白β链(β-TUB)、微管蛋白β-5链(β-TUB5)、微管蛋白α-3链(α-TUB3)、磷酸烯醇丙酮酸羧化酶(PEPC)、酰基载体蛋白2(ACP2)、酰基载体蛋白3(ACP3)。运用Primer Premier 5设计引物,简单克隆和熔解曲线验证引物特异性; 利用qRT-PCR技术检测各个候选内参基因的表达情况,结合GeNorm、NormFinder、BestKeeper软件和RefFinder在线工具综合评估其表达稳定性,并通过目的基因TFL1的表达分析验证其可靠性。结果表明:(1)8个候选内参基因条带位置正确,熔解曲线呈单一峰,说明引物特异性良好。(2)β-TUB、β-TUB5和α-TUB3是‘红元宝'不同花芽分化时期较为稳定的内参基因,而UBC和ACT为稳定性最低的内参基因。(3)β-TUB5、α-TUB3、β-TUB及其组合的相对表达量趋于一致,而ACT和UBC并未对目的基因的表达量进行有效的标准化。因此,β-TUB、β-TUB5和α-TUB3可作为‘红元宝'二次花芽分化研究中稳定表达的内参基因。该研究结果将为木兰属植物二次成花分子调控机制研究提供依据。

Abstract:

A number of Magnolia (Magnoliaceae)species bloom twice each year, instead of once in most other species in this family, which is a desirable ornamental trait. To investigate the molecular mechanism of the flower bud differentiation during the second bloom each year in these Magnolia species, quantitative real-time PCR(qRT-PCR)is frequently used as a sensitive gene expression technique that relies on the stability of reference genes for data normalization. In order to screen the identification of reference gene(s)suitable for molecular characterization of flower bud differentiation stages during second bloom of M. liliflora ‘Hongyuanbao', in this study, leaf and flower bud tissues of M. liliflora ‘Hongyuanbao' at different flower bud differentiation stages were used as materials. Based on transcriptomic sequencing data, eight constitutively expressed genes, including UBC(ubiquitin-conjugating enzyme), ACT(actin), β-TUB(tubulin beta), β-TUB5(tubulin beta), α-TUB3(tubulin alpha), PEPC (phosphoenolpyruvate carboxylase), ACP2(acyl carrier protein 2), ACP3(acyl carrier protein 3), were selected as candidate reference genes for qRT-PCR. Primer Premier 5 was used to design the primers. PCR products of all the eight candidate reference genes were analyzed by gel electrophoresis which showed sharp bands with the expected size. Comprehensively analysis was conducted using four softwares including geNorm, NormFinder, BestKeeper and RefFinder to evaluate its expression stability, and its reliability was verified by expression analysis of the target gene TFL1. The results were as follows:(1)Each melting curve was a single peak, which indicated the high specificity of PCR primers.(2)β-TUB, β-TUB5 and α-TUB3 were the most stable reference genes, whereas UBC and ACT were the lest stable.(3)The relative expressions of β-TUB5, α-TUB3, β-TUB and their combinations showed highly consistent results, while ACT and UBC did not effectively standardize the expression level of TFL1.Therefore, β-TUB5, α-TUB3 and β-TUB can be identified as the most suitable reference genes for qRT-PCR analysis of gene expression in flower bud differentiation during the second bloom of M. liliflora ‘Hongyuanbao'. This study provides useful references for investigating the regulatory mechanisms in Magnolia species flowering.