Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis
Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis
复制标题
坛紫菜应激反应过程中实时 PCR 分析潜在内参基因的鉴定
DOI：
10.1007/s00343-017-6005-9
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发表时间：
2017
影响因子：
--
通讯作者：
Wang Guangce
中科院分区：
文献类型：
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作者：
Wang Xia;Feng Jianhua;Huang Aiyou;He Linwen;Niu Jianfeng;Wang Guangce
坛Pyropia haitanensis是我国特有植物，具有显著的抗逆性。对这些藻类的胁迫响应进行研究，可以为潮间带红藻的胁迫响应机制提供有价值的信息。在这里，盐度和光照强度对光系统II的量子产率的影响在Py。用脉冲幅度调制荧光法研究了坛紫菜的荧光光谱。提取不同胁迫条件下样品的总RNA和基因组DNA。通过对基因组DNA量进行标准化，评价每个样品中的RNA含量。合成cDNA，并使用qRT-PCR方法评价7个潜在内部对照基因的表达水平。然后，我们使用geNorm，一种常用的统计算法，分析七个参考基因的qRT-PCR数据。选择可能在不同条件下持续表达的潜在基因，并且这些基因在盐处理的样品中显示稳定的表达水平，而微管蛋白、甘油醛-3-磷酸脱氢酶和肌动蛋白在强光胁迫的样品中显示稳定性。根据脉冲调幅荧光测定法的结果，进行绝对定量以获得某些应激处理样品中的基因拷贝数。在某些样品中通过绝对定量确定的稳定表达的基因与geNorm筛选的结果一致。根据软件分析和绝对定量的结果，我们提出延伸因子3和18 S核糖体RNA可以作为Py.结果表明，α-微管蛋白和18 S核糖体RNA可作为强光胁迫下的内参基因。在一般情况下，我们的研究结果提供了一个方便的参考选择的内部控制基因时，设计实验相关的压力反应在Py。Haitanensis。
Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde-3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that α-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.