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用于……中定量实时PCR的合适内参基因的选择

Selection of Suitable Reference Genes for Quantitative Real-time PCR in .

作者信息

Chen Xue, Mao Yingji, Huang Shengwei, Ni Jun, Lu Weili, Hou Jinyan, Wang Yuting, Zhao Weiwei, Li Minghao, Wang Qiaojian, Wu Lifang

机构信息

Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of SciencesHefei, China.

Institute of Technical Biology & Agriculture Engineering, Science Island Branch of Graduate School, University of Science and Technology of ChinaHefei, China.

出版信息

Front Plant Sci. 2017 May 4;8:637. doi: 10.3389/fpls.2017.00637. eCollection 2017.

Abstract

Chinese tallow ( L.) is a promising landscape and bioenergy plant. Measuring gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) can provide valuable information on gene function. Stably expressed reference genes for normalization are a prerequisite for ensuring the accuracy of the target gene expression level among different samples. However, the reference genes in Chinese tallow have not been systematically validated. In this study, 12 candidate reference genes (, and ) were investigated with qRT-PCR in 18 samples, including those from different tissues, from plants treated with sucrose and cold stresses. The data were calculated with four common algorithms, geNorm, BestKeeper, NormFinder, and the delta cycle threshold (ΔCt). and were the most stable for the tissue-specific experiment, and for cold treatment, and and for sucrose stresses, while the least stable genes were , and respectively. The comprehensive results showed , and to be the top-ranked stable genes across all the samples. The stability of was the lowest during all experiments. These selected reference genes were further validated by comparing the expression profiles of the chalcone synthase gene in Chinese tallow in different samples. The results will help to improve the accuracy of gene expression studies in Chinese tallow.

摘要

乌桕(Sapium sebiferum (L.) Roxb.)是一种很有前景的景观和生物能源植物。通过定量实时聚合酶链反应(qRT-PCR)测量基因表达可以提供有关基因功能的有价值信息。用于标准化的稳定表达的内参基因是确保不同样本间靶基因表达水平准确性的前提条件。然而,乌桕中的内参基因尚未得到系统验证。在本研究中,利用qRT-PCR对12个候选内参基因(ACT、UBQ、EF1α、TUA、TUB、GAPDH、β-TUB、18S rRNA、SAMDC、PP2A、G6PDH和UBC)在18个样本中进行了研究,这些样本包括来自不同组织的样本,以及经蔗糖和冷胁迫处理的植株的样本。数据采用四种常用算法geNorm、BestKeeper、NormFinder和ΔCt进行计算。对于组织特异性实验,ACT和UBQ最稳定;对于冷处理,EF1α和TUA最稳定;对于蔗糖胁迫,GAPDH和β-TUB最稳定,而最不稳定的基因分别是18S rRNA、SAMDC和UBC。综合结果表明,在所有样本中,ACT、UBQ和EF1α是排名最靠前的稳定基因。在所有实验中,18S rRNA的稳定性最低。通过比较乌桕中查尔酮合酶基因在不同样本中的表达谱,对这些选定的内参基因进行了进一步验证。研究结果将有助于提高乌桕基因表达研究的准确性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f0b3/5415600/c292b0c28bb0/fpls-08-00637-g0001.jpg

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