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Validation of reference genes for quantitative RT-PCR normalization in Suaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy.

ABSTRACT: Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful analytical technique for the measurement of gene expression, which depends on the stability of the reference gene used for data normalization. Suaeda aralocaspica, an annual halophyte with heteromorphic seeds and possessing C4 photosynthesis pathway without Kranz anatomy, is an ideal plant species to identify stress tolerance-related genes and compare relative expression at transcriptional level. So far, no molecular information is available for this species. In the present study, six traditionally used reference genes were selected and their expression stability in two types of seeds of S. aralocaspica under different experimental conditions was evaluated. Three analytical programs, geNorm, NormFinder and BestKeeper, were used to assess and rank the stability of reference gene expression. Results revealed that although some reference genes may display different transcriptional profiles between the two types of seeds, ?-TUB and GAPDH appeared to be the most suitable references under different developmental stages and tissues. GAPDH was the appropriate reference gene under different germination time points and salt stress conditions, and ACTIN was suitable for various abiotic stress treatments for the two types of seeds. For all the sample pools, ?-TUB served as the most stable reference gene, whereas 18S rRNA and 28S rRNA performed poorly and presented as the least stable genes in our study. UBQ seemed to be unsuitable as internal control under different salt treatments. In addition, the expression of a photosynthesis-related gene (PPDK) of C4 pathway and a salt tolerance-related gene (SAT) of S. aralocaspica were used to validate the best performance reference genes. This is the first systematic comparison of reference gene selection for qRT-PCR work in S. aralocaspica and these data will facilitate further studies on gene expression in this species and other euhalophytes.

SUBMITTER: Cao J

PROVIDER: S-EPMC4756755 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Validation of reference genes for quantitative RT-PCR normalization in Suaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy.

Cao Jing J Wang Lu L Lan Haiyan H

PeerJ 20160211

Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful analytical technique for the measurement of gene expression, which depends on the stability of the reference gene used for data normalization. Suaeda aralocaspica, an annual halophyte with heteromorphic seeds and possessing C4 photosynthesis pathway without Kranz anatomy, is an ideal plant species to identify stress tolerance-related genes and compare relative expression at transcriptional level. So fa ...[more]

PMID: 26893974

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Project description:Phosphoenolpyruvate carboxylase (PEPC) plays pivotal roles in the carbon fixation of photosynthesis and a variety of metabolic and stress pathways. Suaeda aralocaspica belongs to a single-cellular C4 species and carries out a photosynthetic pathway in an unusually elongated chlorenchyma cell, which is expected to have PEPCs with different characteristics. To identify the different isoforms of PEPC genes in S. aralocaspica and comparatively analyze their expression and regulation patterns as well as the biochemical and enzymatic properties in this study, we characterized a bacterial-type PEPC (BTPC; SaPEPC-4) in addition to the two plant-type PEPCs (PTPCs; SaPEPC-1 and SaPEPC-2) using a genome-wide identification. SaPEPC-4 presented a lower expression level in all test combinations with an unknown function; two SaPTPCs showed distinct subcellular localizations and different spatiotemporal expression patterns but positively responded to abiotic stresses. Compared to SaPEPC-2, the expression of SaPEPC-1 specifically in chlorenchyma cell tissues was much more active with the progression of development and under various stresses, particularly sensitive to light, implying the involvement of SaPEPC-1 in a C4 photosynthetic pathway. In contrast, SaPEPC-2 was more like a non-photosynthetic PEPC. The expression trends of two SaPTPCs in response to light, development, and abiotic stresses were also matched with the changes in PEPC activity in vivo (native) or in vitro (recombinant), and the biochemical properties of the two recombinant SaPTPCs were similar in response to various effectors while the catalytic efficiency, substrate affinity, and enzyme activity of SaPEPC-2 were higher than that of SaPEPC-1 in vitro. All the different properties between these two SaPTPCs might be involved in transcriptional (e.g., specific cis-elements), posttranscriptional [e.g., 5'-untranslated region (5'-UTR) secondary structure], or translational (e.g., PEPC phosphorylation/dephosphorylation) regulatory events. The comparative studies on the different isoforms of the PEPC gene family in S. aralocaspica may help to decipher their exact role in C4 photosynthesis, plant growth/development, and stress resistance.

Dataset Information

Validation of reference genes for quantitative RT-PCR normalization in Suaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy.

Publications

Validation of reference genes for quantitative RT-PCR normalization in Suaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy.

Similar Datasets

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