Assessment of Appropriate Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Normalisation in Magnolia sieboldii K. Koch across Various Experimental Conditions
Assessment of Appropriate Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Normalisation in Magnolia sieboldii K. Koch across Various Experimental Conditions
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DOI：
10.15244/pjoes/143508
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发表时间：
2022-02
影响因子：
1.8
通讯作者：
Shixin Guan;Shi Qiu;M. Mei;Xin Hao;Xiaolin Zhang;Xiu-jun Lu
中科院分区：
文献类型：
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作者：
Shixin Guan;Shi Qiu;M. Mei;Xin Hao;Xiaolin Zhang;Xiu-jun Lu
天女木兰科赫是一种著名的观赏植物。种子的深度休眠使育种困难，并阻止了经济目的的种植。萌发相关基因的表达谱研究可为揭示打破休眠的分子机制提供线索。通过定量实时聚合酶链反应（RT-qPCR）（开发用于研究基因表达谱）获得的结果的准确性取决于所选参考基因的适用性。然而，尚未在M中确定合适的RT-qPCR参考基因。西氏菌在本研究中，7个潜在的参考基因在不同的M。通过RT-qPCR检查不同器官组织、种子发育阶段和经历吸胀和层积处理的种子的天女花样品亚组。通过三种统计算法，即geNorm、Normalization和BestKeeper，评估和分析基因表达稳定性。亲环素（CYC）和泛素结合酶（UCE）被确定为不同器官的最佳参考基因。CYC和UCE在不同发育时期的种子中被评估为合适的参考基因。铜锌超氧化物歧化酶（Cu/Zn-SOD）和锰超氧化物歧化酶（Mn-SOD）是种子吸胀处理的最佳参考基因，而Cu/Zn-SOD和30S核糖体蛋白S13（RPS13）是种子层积处理的最佳参考基因。in m.因此，推荐Cu/Zn-SOD用于RT-qPCR标准化，而DBP不适合用于基因表达分析。为了更好地验证参考基因的可信度，分析了DELAY OF GERMINATION 1（DOG 1）的表达模式。本研究提供 * 电子邮件：www.example.com ** 电子邮件：www.example.com DOI：10.15244/pjoes/143508在线出版日期：
Magnolia sieboldii K. Koch is a famous ornamental plant. The deep dormancy of the seeds makes breeding difficult and prevents cultivation for economic purposes. Gene expression profiles of germination-related genes can provide clues for understanding the molecular mechanism of dormancy breaking. The accuracy of results acquired through quantitative real-time polymerase chain reaction (RT-qPCR), which was developed for investigating the gene expression profiles, is determined by the suitability of the selected reference gene. Nevertheless, appropriate RT-qPCR reference genes have not been determined in M. sieboldii. In the present study, seven potential reference genes in diverse M. sieboldii sample subgroups of different organ tissues, seed developmental stages, and seed subjected to imbibition and stratification treatments were checked through RT-qPCR. The gene expression stability was assessed and analysed through three statistical algorithms, namely, geNorm, NormFinder, and BestKeeper. Cyclophilin (CYC) and ubiquitin-conjugating enzyme (UCE) were identified as the best reference genes for diverse organs. CYC and UCE were evaluated as suitable reference genes in seeds in diverse periods of development. Copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) served as optimal reference genes during seed imbibition treatment, whereas Cu/Zn-SOD and 30S ribosomal protein S13 (RPS13) were optimal during seed stratification treatment. In M. sieboldii, Cu/Zn-SOD was recommended for RT-qPCR normalisation, whereas DBP was inappropriate for gene expression analysis. The expression pattern of DELAY OF GERMINATION1 (DOG1) was analysed for validating reference gene creditability better. The present study provides *e-mail: zhanglin_25683921@hotmail.com **e-mail: lxjsyau@syau.edu.cn DOI: 10.15244/pjoes/143508 ONLINE PUBLICATION DATE: