Selection of reference genes for quantitative real-time PCR in Casuarina equisetifolia under salt stress
Selection of reference genes for quantitative real-time PCR in Casuarina equisetifolia under salt stress
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DOI：
10.1007/s10535-016-0670-y
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发表时间：
2017-09-01
影响因子：
1.5
通讯作者：
Guo, G.
中科院分区：
文献类型：
--
作者：
Fan, C.;Qiu, Z.;Guo, G.
实时定量PCR （Real - time quantitative PCR, qPCR）以其准确性和敏感性被广泛应用于基因表达分析。内参基因作为内源控制基因的正常化是必要的。为了选择合适的内参基因来规范盐胁迫下木麻黄（Casuarina equisetifolia）基因的表达，采用real - time qPCR技术对不同NaCl浓度和处理时间下木麻黄叶片和根系中的10个潜在内参基因进行了筛选。GeNorm、NormFinder和BestKeeper分析显示，伸长因子1- α (EF1 α)和泛素偶联酶E2 （UBC）是盐胁迫下实时qPCR最合适的内参基因。然而，在其他植物物种中被广泛用作内参基因的β -微管蛋白（β - TUB）和肌动蛋白7的表达并不稳定。EF1 α、UBC、未表征蛋白2、DNAJ同源亚家族A成员2和甘油醛-3-磷酸脱氢酶的组合应该是盐胁迫下所有样品中基因表达数据正常化的理想参考基因。这表明，为了使马叶松基因表达正常化，需要选择内参基因。此外，通过验证WRKY29-like和expansin-like B1的表达，确认所选内参基因的适宜性。研究结果可用于分析马叶松对盐的响应机制和基因表达。
Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1 alpha) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, beta-tubulin (beta TUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1 alpha, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.